Treatment of interstitial cystitis

ABSTRACT

The present invention relates to the use of an anti-NGF antibody in the treatment or prevention of pain and/or a lower urinary tract symptom (LUTS) associated with interstitial cystitis and/or painful bladder syndrome and/or bladder pain syndrome.

This application is a national stage filing of PCT/IB2008/055383 filedDec. 17, 2008, which claims the benefit of Provisional PatentApplication No. 61/061,149 filed Jun. 13, 2008, and the benefit ofProvisional Patent Application No. 61/014,171 filed Dec. 17, 2007.

FIELD OF THE INVENTION

The present invention relates to the use of an anti-NGF antibody in thetreatment or prevention of pain and/or a lower urinary tract symptom(LUTS) associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome.

BACKGROUND OF THE INVENTION

Interstitial cystitis (IC) is a chronic bladder disease, of unknownorigin, characterized by symptoms of pain, such as pelvic pain, andlower urinary tract symptoms (LUTS) such as increased urinaryfrequency/urgency. More recently terminology has evolved to includepainful bladder syndrome (PBS) (MacDiarmid S A et al. Rev Urol 2007;9(1): 9-16) or bladder pain syndrome (BPS) (van der Merve et al.European Urology 53(2008) 60-67, along with IC, that is IC/PBS/BPS tocollectively describe this symptom complex.

Prevalence rates of IC/PBS/BPS vary from 67 to 230 per 100,000 womenhaving clinically confirmed disease, although the number is likelyhigher than this due to under- or mis-diagnosis, commonly asendometriosis, recurrent urinary tract infection, overactive bladder orvulvodynia (Forrest J B et al. Clinical Courier 2006; 24(3): 1-8). IChas a significant impact on quality of life, affecting travel, familyrelationships, and employment (Slade D et al. Urol 1997; 49 (5A Suppl):10-3), as well as being associated with depressive symptoms (Rothrock NE et al. J Urol 2002; 167: 1763-1767).

There are few well performed, placebo-controlled, randomized trials oftherapies aimed at IC, and treatment often consists of a multimodaltrial-and-error approach, as evidenced by one review of patients in theInterstitial Cystitis Data Base study which reported 183 different typesof treatment (Rovner E et al. J Urol 2000; 56: 940-5).

No single etiology has been identified, and it is most likely amultifactorial process with several urologic insults causing aself-perpetuating process of epithelial cell dysfunction, C-nerve fibreactivation, and proliferation of mast cells, leading to worsening tissuedamage, scarring and fibrosis. The repetitive stimulation of C fibresfrom inflammation, and upregulation of sensory nerves in the bladder,ultimately leads to permanent alterations (centralization) resulting inhyperalgesia, chronic bladder pain and voiding dysfunction (Forrest J Bet al. Clinical Courier 2006; 24(3):1-8).

Although no consensus has been reached on the fundamental causes of IC,existing data have led to speculation that three pathophysiologicmechanisms may be implicated: epithelial dysfunction, mast cellactivation, and neurogenic inflammation (Nazif O et al. Urol 2007; 69(Suppl 4A): 24-33).

There is a continuing need to provide a novel, effective treatment forpain and/or a lower urinary tract symptom of interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome without theadverse effects or limited efficacy of currently available therapies.

There has been a report that a recombinant human anti-NGF (nerve growthfactor) antibody has been tested in patients with interstitial cystitis(Dimitrakov, et al., J. Urology. Vol 171(4), Supplement, 363 (2004)).Although the objective of the study was to evaluate the efficacy andsafety “in a group of IC patients with significant nerve damage andneuropathic pain component who have failed numerous previoustreatments”, only effects on nerve damage were reported using urinarynerve damage marker levels. It is important to note that no efficacy isreported in treating pain and/or a lower urinary tract symptom of thecondition.

The anti-NGF antibody E3 has previously been reported as being useful inthe treatment of pain, including rheumatoid arthritis pain,osteoarthritis pain and post-surgical pain (see for exampleWO2004/058184). However, given the etiology of interstitial cystitis andpoor mechanistic understanding of the condition, it cannot be easilypredicted that an anti-NGF antibody, such as antibody E3, could be usedfor treating pain and/or a lower urinary tract symptom (LUTS) associatedwith interstitial cystitis and/or painful bladder syndrome and/orbladder pain syndrome.

BRIEF SUMMARY OF THE INVENTION

In an aspect of the invention there is provided a method for treating orpreventing pain and/or a lower urinary tract symptom (LUTS) associatedwith interstitial cystitis and/or painful bladder syndrome and/orbladder pain syndrome, comprising administering an effective amount ofan anti-NGF antagonist antibody. In one embodiment, the antibody:

(a) binds NGF with a K_(D) of less than about 2 nM;

(b) inhibits human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 100 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM human NGF; and/or

(c) inhibits human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 10 pM or less, wherein the IC50 ismeasured in the presence of about 1.5 pM of NGF.

Pain associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome comprise lower abdominal (pelvic)pain; bladder pain; suprapubic pain; vaginal pain; pain in the penis,testicles, scrotum and perineum; urethral pain; dyspareneuria; pain,pressure or discomfort that may increase as the bladder fills.

Lower urinary tract symptoms comprise three groups of urinary symptoms,which may be defined as storage (irritative), voiding (obstructive) andpost-micturition symptoms. Storage symptoms comprise urgency, frequency,nocturia, urgency incontinence and stress incontinence, which can beassociated with overactive bladder (OAB) and benign prostatichyperplasia (BPH). Voiding symptoms comprise hesitancy, poor flow,intermittency, straining and dysuria. Post-micturition symptoms compriseterminal dribbling, post-void dribbling and a sense of incompleteemptying.

Over Active Bladder (OAB) is defined as urgency, with or without urgeincontinence, usually with frequency and nocturia [Abrams et al.,Neurourology and Urodynamics 21:167-178 (2002)]. Prevalence of OAB inmen and women is similar, with approximately 16% of the population ofthe USA suffering from the condition [Stewart et al, Prevalence ofOveractive Bladder in the United States: Results from the NOBLE Program;Abstract Presented at the 2^(nd) International Consultation onIncontinence, July 2001, Paris, France].

The terms OAB Wet and OAB Dry describe OAB patients with or withouturinary incontinence respectively. Previously, the cardinal symptom ofOAB was believed to be urinary incontinence. However, with the advent ofthe new terms this is clearly not meaningful for the large number ofsufferers who are not incontinent (i.e. OAB Dry patients). Thus, a 2001study from Liberman et al [‘Health Related Quality of Life Among Adultswith Symptoms of Overactive Bladder: Results From A US Community-BasedSurvey’; Urology 57(6), 1044-1050, 2001] examined the impact of all OABsymptoms on the quality of life of a community-based sample of the USpopulation. This study demonstrated that individuals suffering from OABwithout any demonstrable loss of urine have an impaired quality of lifewhen compared with controls.

BPH is a chronically progressive disease that can lead to complicationssuch as acute urinary retention, recurrent urinary tract infections,bladder stones and renal dysfunction. The prevalence and averageseverity of LUTS associated with BPH in men increases with age.

BPH leads to an increase in prostate volume, creating urethral andbladder outflow obstruction as well as secondary changes in bladderfunction. The effects of this are manifested by both storage(irritative) and voiding (obstructive) symptoms.

It has been shown in accordance with the invention that an anti-NGFantagonist antibody is capable of inhibiting or blocking pain and/or alower urinary tract symptom (LUTS) associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome. In someembodiments, the pain and/or lower urinary tract symptom is/arealleviated within about 24 hours after administering the anti-NGFantagonist antibody. In some embodiments, the pain and/or lower urinarytract symptom is/are alleviated within about 4 days after administeringthe anti-NGF antagonist antibody. In some embodiments, the pain and/orlower urinary tract symptom is/are alleviated before observing or in theabsence of an indication of improvement of the condition in theindividual.

In a further aspect of the invention there is provided a method fortreating or preventing pain and/or a lower urinary tract symptom (LUTS)associated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome, comprising administering an effectiveamount of an anti-NGF antagonist antibody comprising a heavy chainvariable region comprising:

(a) a CDR1 region shown in SEQ ID NO: 3;

(b) a CDR2 region shown in SEQ ID NO:4; and

(c) a CDR3 region shown in SEQ ID NO:5.

In a further aspect of the invention there is provided a method fortreating or preventing pain and/or a lower urinary tract symptom (LUTS)associated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome, comprising administering an effectiveamount of an anti-NGF antagonist antibody comprising a light chainvariable region comprising:

(a) a CDR1 region shown in SEQ ID NO: 6;

(b) a CDR2 region shown in SEQ ID NO:7; and

(c) a CDR3 region shown in SEQ ID NO:8.

The anti-NGF antagonist antibody may further comprise a heavy chainvariable region comprising:

(a) a CDR1 region shown in SEQ ID NO: 3;

(b) a CDR2 region shown in SEQ ID NO:4; and

(c) a CDR3 region shown in SEQ ID NO:5.

The anti-NGF antagonist antibody may comprise a heavy chain variableregion comprising an amino acid sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID No. 1 and a light chain variable region comprising anamino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID No.2, wherein the antibody binds specifically to NGF.

The heavy chain variable region and/or light chain variable region ofthe anti-NGF antibody may comprise one or more respective frameworkmutations. In one aspect the framework mutation may replace a humanframework residue with the complementary murine framework residue. Themutation may comprise the substitution V71K in the heavy chain variableregion.

The anti-NGF antagonist antibody may comprise a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 1 and/or maycomprise a light chain variable region comprising the amino acidsequence of SEQ ID NO: 2.

The anti-NGF antagonist antibody may be an antibody comprising the aminoacid sequences shown in SEQ ID NOS: 1 and 2. The anti-NGF antagonistantibody may be an antibody comprising the amino acid sequences shown inSEQ ID NOS: 16 and 17.

The anti-NGF antagonist antibody may comprise a heavy chain comprisingan amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID No.16 and a light chain comprising an amino acid sequence at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID No. 17, wherein the antibody bindsspecifically to NGF.

The anti-NGF antagonist antibody may compete for NGF binding with ananti-NGF antagonist antibody as defined herein. The anti-NGF antagonistantibody may compete for NGF binding with an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ ID NO: 1and a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 2.

The anti-NGF antagonist antibody may be a humanized. The antibody may beantibody E3, which specifically binds human and rodent NGF. Antibody E3is described in WO2004/058184, the content of which is herebyincorporated by reference in its entirety. The amino acid sequences ofthe heavy chain and light chain variable regions of E3 are shown in SEQID Nos. 1 and 2 (FIGS. 1A and 1B of WO2004/058184), respectively. TheCDR portions of antibody E3 (including Chothia and Kabat CDRs) arediagrammatically depicted in FIGS. 1A and 1B of WO2004/058184. The aminoacid sequences of E3 heavy and light chains, and of the individualextended CDRs are also shown below (See, “antibody sequences”, below).Antibody E3 is highly potent in sequestering NGF and preventinginteraction with its receptor. E3 and its murine precursor antibody 911have been shown to be an effective analgesic in non-clinical animalmodels of pathological pain including arthritis, cancer pain and postsurgical pain.

In another aspect, the antibody comprises a fragment or a region of theantibody E3 (interchangeably termed “E3” herein). In one embodiment, thefragment is a light chain of the antibody E3 as shown in FIG. 1B ofWO2004/058184 and SEQ ID No. 17 herein. In another embodiment, thefragment is a heavy chain of the antibody E3 as shown in FIG. 1A ofWO2004/058184 and SEQ ID No. 16 herein. In yet another embodiment, thefragment contains one or more variable regions from a light chain and/ora heavy chain of the antibody E3. In yet another embodiment, thefragment contains one or more CDRs from a light chain and/or a heavychain of the antibody E3 as shown in FIGS. 1A and 1B of WO2004/058184and SEQ ID Nos. 17 and 16, respectively, herein.

In another aspect, the antibody comprises a light chain that is encodedby a polynucleotide that is produced by a host cell with a depositnumber of ATCC No. PTA-4893 or ATCC No. PTA-4894. In another aspect, theantibody comprises a heavy chain that is encoded by a polynucleotidethat is produced by a host cell with a deposit number of ATCC No.PTA-4895. In another aspect, the antibody comprises (a) a light chainthat is encoded by a polynucleotide that is produced by a host cell witha deposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893; and (b) aheavy chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4895 (for convenienceherein, the polynucleotide(s) produced by a deposited host cell arereferred to as having a deposit number of ATCC NOs PTA-4894, PTA-4893and PTA-4895). In another aspect, the antibody comprises a light chainvariable region of a light chain that is encoded by a polynucleotidethat is produced by a host cell with a deposit number of ATCC No.PTA-4894 or ATCC No. PTA-4893. In another aspect, the antibody comprisesa heavy chain variable region of a heavy chain that that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4895. In another aspect, the antibody comprises (a) a lightchain variable region of a light chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894 or ATCC No. PTA-4893, and (b) a heavy chain variableregion of a heavy chain that that is encoded by a polynucleotide that isproduced by a host cell with a deposit number of ATCC No. PTA-4895. Instill another aspect, the antibody comprises one or more CDR(s) encodedby (a) a polynucleotide that is produced by a host cell with a depositnumber of ATCC No. PTA-4894; and/or (b) a heavy chain that is encoded bya polynucleotide that is produced by a host cell with a deposit numberof ATCC No. PTA-4895.

In some embodiments, the antibody may be a full length antibody, whichmay be of the IgG2 subclass. The antibody may comprise the human heavychain IgG2a constant region. In some embodiments the antibody comprisesthe human light chain kappa constant region. In some embodiments, theantibody comprises a modified constant region, such as a constant regionthat is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). In other embodiments, the constant region ismodified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCTWO9958572. The antibody may comprise a human heavy chain IgG2a constantregion comprising the following mutations: A330P331 to S330S331 (aminoacid numbering with reference to the wildtype IgG2a sequence).

In some embodiments, the antibody may be an antibody fragment, such asFab or Fab′2 fragments, or a single chain antibody.

In another aspect, the antibody comprises any one or more of thefollowing: a) one or more CDR(s) of antibody E3 shown in SEQ ID Nos. 1-8(FIGS. 1A and 1B of WO2004/058184); b) CDR H3 from the heavy chain ofantibody E3 shown in SEQ ID No. 1 and 5 (FIG. 1A of WO2004/058184); c)CDR L3 from the light chain of antibody E3 shown in SEQ ID No. 2 and 8(FIG. 1B of WO2004/058184); d) three CDRs from the light chain ofantibody E3 shown in SEQ ID No. 2, 6-8 (FIG. 1B of WO2004/058184); e)three CDRs from the heavy chain of antibody E3 shown in SEQ ID Nos. 1,3-5 (FIG. 1A of WO2004/058184); and f) three CDRs from the light chainand three CDRs from the heavy chain, of antibody E3 shown in SEQ ID Nos.1-8 (FIGS. 1A and 1B of WO2004/058184). In another aspect, the antibodymay comprise any one or more of the following: a) one or more (one, two,three, four, five, or six) CDR(s) derived from antibody E3 shown in SEQID Nos. 1-8 (FIGS. 1A and 1B of WO2004/058184); b) a CDR derived fromCDR H3 from the heavy chain of antibody E3 shown in SEQ ID Nos. 1 and 5(FIG. 1A of WO2004/058184); and/or c) a CDR derived from CDR L3 from thelight chain of antibody E3 shown in SEQ ID Nos. 2 and 8 (FIG. 1B ofWO2004/058184). In some embodiments, the CDRs may be Kabat CDRs, ChothiaCDRs, or a combination of Kabat and Chothia CDRs (termed “extended” or“combined” CDRs herein). In some embodiments, the polypeptides compriseany of the CDR configurations (including combinations, variants, etc.)described herein.

In one aspect, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 issubstituted with N, T or S. For convenience herein, “substituted” or“is” in this context or reference to an amino acid refers to choices ofamino acid(s) for a given position. As is clear, the substitution, orchoice, may be the amino acid depicted in a SEQ ID herein.

In another aspect, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A,or S; and L63 is L or V.

In another aspect, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Yor W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Y110 isY, K, S, R or T.

In another aspect, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Yor W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; andwherein Y110 is any amino acid.

In another aspect, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D, or T;wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid.

In another aspect, the antibody comprises a light chain variable regioncomprising SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y;and H32 is H, N, or Q.

In another aspect, the antibody comprises a light chain variable regioncomprising SEQ ID NO: 13, wherein I51 is I, T, V or A; and S56 is S orT.

In another aspect, the antibody comprises a light chain variable regioncomprising SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, or S;and wherein Y96 is Y or R.

In another aspect, the antibody comprises a light chain variable regioncomprising SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid;T93 is any amino acid; and wherein Y96 is Y or R.

In one aspect, the antibody comprises an amino acid sequence shown inSEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 is N, T or S.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; andL63 is L or V.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S,R or T.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and whereinY110 is any amino acid.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G;and wherein Y110 is any amino acid.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 isH, N, or Q.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO: 13, wherein I51 is I, T, V or A; and S56 is S or T.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; andwherein Y96 is Y or R.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 isany amino acid; and wherein Y96 is Y or R.

In another aspect, the antibody comprises a heavy chain variable regioncomprising the CDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A, orI; and N35 is N, T or S; the CDR2 region of SEQ ID NO:10, wherein M50 isM, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3region of SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y orW; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 isS, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y,K, S, R or T. In some embodiments, the heavy chain variable regioncomprises the CDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; wherein Y110 is any amino acid. In other embodiments, theheavy chain variable region comprises the CDR3 region of SEQ ID NO:11,wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V,N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid. In some embodiments, antibody further comprises an antibodylight chain variable region.

In another aspect, the antibody comprises a light chain variable regioncomprising the CDR1 region of SEQ ID NO:12, wherein S26 is S or F; D28is D, S, A, or Y; and H32 is H, N, or Q; the CDR2 region of SEQ IDNO:13, wherein I51 is I, T, V or A; and S56 is S or T; and the CDR3region of SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; andwherein Y96 is Y or R. In some embodiments, the light chain variableregion comprises the CDR3 region of SEQ ID NO:14, wherein S91 is S or E;K92 is any amino acid; T93 is any amino acid; and wherein Y96 is Y or R.In some embodiments, the antibody further comprises an antibody heavychain.

In another aspect, the antibody comprises (a) a heavy chain variableregion comprising the CDR1 region of SEQ ID NO:9, wherein I34 is S, L, VA, or I; and N35 is N, T or S; the CDR2 region of SEQ ID NO:10, whereinM50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and theCDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 isY or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y,K, S, R or T; and (b) a light chain variable region comprising the CDR1region of SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; andH32 is H, N, or Q; the CDR2 region of SEQ ID NO:13, wherein I51 is I, T,V or A; and S56 is S or T; and the CDR3 region of SEQ ID NO:14, whereinS91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the light chain variable region comprises the CDR3 regionof SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 isany amino acid; and wherein Y96 is Y or R. In some embodiments, theheavy chain variable region comprises the CDR3 region of SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the heavy chain variable region comprisesthe CDR3 region of SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, theantibody further comprises an antibody light chain.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 is N, T or S; anamino acid sequence shown in SEQ ID NO:10, wherein M50 is M, I, G, Q, S,or L; A62 is A, or S; and L63 is L or V; and an amino acid sequenceshown in SEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y orW; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 isS, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y,K, S, R or T. In some embodiments, the antibody comprises an amino acidsequence shown in SEQ ID NO:11, wherein Y100 is Y, L, or R; and whereinY101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S;wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T,or G; and wherein Y110 is any amino acid. In other embodiments, thepolypeptide comprises an amino acid sequence shown in SEQ ID NO:11,wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V,N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid. In some embodiments, the antibody further comprises anantibody light chain variable region.

In another aspect, the antibody comprises an amino acid sequence shownin SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 isH, N, or Q; an amino acid sequence shown in SEQ ID NO:13, wherein I51 isI, T, V or A; and S56 is S or T; and an amino acid sequence shown in SEQID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96is Y or R. In some embodiments, the antibody comprises an amino acidsequence shown in SEQ ID NO:14, wherein S91 is S or E; K92 is any aminoacid; T93 is any amino acid; and wherein Y96 is Y or R. In someembodiments, the antibody further comprises an antibody heavy chainvariable region.

In another aspect, the antibody comprises (a) an amino acid sequenceshown in SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 is N, T orS; an amino acid sequence shown in SEQ ID NO:10, wherein M50 is M, I, G,Q, S, or L; A62 is A, or S; and L63 is L or V; and an amino acidsequence shown in SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Y110is Y, K, S, R or T; and (b) an amino acid sequence shown in SEQ IDNO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, orQ; an amino acid sequence shown in SEQ ID NO:13, wherein I51 is I, T, Vor A; and S56 is S or T; and an amino acid sequence shown in SEQ IDNO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Yor R. In some embodiments, the antibody comprises an amino acid sequenceshown in SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93is any amino acid; and wherein Y96 is Y or R. In some embodiments, theantibody comprises an amino acid sequence shown in SEQ ID NO:11, whereinY100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; wherein Y110 is any amino acid. In otherembodiments, the polypeptide comprises an amino acid sequence shown inSEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 isG, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Yor W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; andwherein Y110 is any amino acid. In some embodiments, the antibodyfurther comprises an antibody light chain variable region.

In another aspect, the antibody comprises a heavy chain variable regioncomprising: (a) a CDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A,or I; and N35 is substituted with N, T or S; (b) a CDR2 region of SEQ IDNO:10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L orV; and (c) a CDR3 region of SEQ ID NO: 11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; andwherein Y110 is Y, K, S, R or T; wherein the antibody binds NGF.

In another aspect, the antibody comprises a light chain variable regioncomprising: (a) a CDR1 region of SEQ ID NO:12, wherein S26 is S or F;D28 is D, S, A, or Y; and H32 is H, N, or Q; (b) a CDR2 region of SEQ IDNO: 13, wherein I51 is I, T, V or A; and S56 is S or T; and (c) a CDR3region of SEQ ID NO:14, wherein K92 is K, H, R, or S; and wherein Y96 isY or R; wherein the antibody binds NGF.

In another aspect, the antibody comprises (a) a heavy chain variableregion comprising: (i) a CDR1 region of SEQ ID NO:9, wherein I34 issubstituted with S, L, V A, or I; and N35 is substituted with N, T or S;(ii) a CDR2 region of SEQ ID NO:10, wherein M50 is I, G, Q, S, or L; A62is A, or S; and L63 is L or V; and (iii) a CDR3 region of SEQ ID NO: 11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is D, N, or G; wherein Y110 is Y, K, S, R or T; and (b) alight chain variable region comprising: (i) a CDR1 region of SEQ IDNO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, orQ; (ii) a CDR2 region of SEQ ID NO: 13, wherein I51 is I, T, V or A; andS56 is S or T; and (iii) a CDR3 region of SEQ ID NO:14, wherein S91 is Sor E; K92 is K, H, R, or S; and wherein Y96 is Y or R; wherein theantibody binds NGF.

Unless otherwise noted, choice (e.g., substitution) of an amino acid inone location is independently selected from selection of an amino acidin any other location.

In some embodiments, the antibodies comprise any of the CDRconfigurations (including combinations, variations, etc.) describedherein.

As is evident from the description herein, the variable region numberingused herein is sequential numbering. One of skill in the art readilyunderstands that a number of antibody numbering systems exist (such asKabat and Chothia numbering), and how to convert sequential numberinginto another numbering system, such as Kabat numbering or Chothianumbering.

In another aspect, the antibody comprises an amino acid sequence (suchas a CDR3 sequence) selected from SEQ ID NO:46 or 50. In still otherembodiments, the antibody further comprises one or more of the aminoacid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still otherembodiments, the antibody further comprises one of more of the aminoacid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

In another aspect, the antibody comprises an amino acid sequence (suchas a CDR region, such as a CDRH1 and/or CDR H2 region) selected from (a)SEQ ID NOS:28 and/or 29; (b) SEQ ID NOS:30 and/or 31; (c) SEQ ID NOS:32and/or 33; (d) SEQ ID NOS:34 and/or 35; (e) SEQ ID NOS:36 and/or 37; (f)SEQ ID NOS:38 and/or 39; and (g) SEQ ID NOS:40 and 41. In someembodiments, the antibody comprises an amino acid sequence (such as aCDR H1 region) selected from SEQ ID NOS:28, 30, 32, 34, 36, 38, and 40.In some embodiments, the antibody comprises an amino acid sequence (suchas a CDR H2 region) selected from SEQ ID NOS:29, 31, 33, 35, 37, 39 and41. In still other embodiments, the antibody further comprises one ormore of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and8. In still other embodiments, the antibody further comprises one ofmore of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13,14, and 15.

In another aspect, the antibody comprises an amino acid sequence (suchas a CDR region, such as a CDRL1 and/or CDR L2 region) selected from (a)SEQ ID NOS:18 and/or 19; (b) SEQ ID NOS:20 and/or 21; and (c) SEQ IDNOS:22 and/or 23. In some embodiments, the polypeptide comprises anamino acid sequence (such as a CDR L1 region) selected from SEQ IDNOS:18, 20, and 22. In some embodiments, the antibody comprises an aminoacid sequence (such as a CDR L2 region) selected from SEQ ID NOS:19, 21,and 23. In still other embodiments, the antibody further comprises oneor more of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7,8. In still other embodiments, the antibody further comprises one ofmore of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13,14, and 15.

In another aspect, the antibody comprises an amino acid sequence (suchas a CDR region, such as a CDRL3 and/or CDR H3 region) selected from (a)SEQ ID NOS:51 and/or 52; (b) SEQ ID NOS:55 and/or 56; (c) SEQ ID NOS:57and/or 58; (c) SEQ ID NOS:59 and/or 60; (d) SEQ ID NOS:61 and/or 62; (e)SEQ ID NOS:63 and/or 64. In some embodiments, the antibody comprises anamino acid sequence (such as a CDR L3 region) selected from SEQ IDNOS:51, 55, 57, 59, 61, and 63. In some embodiments, the antibodycomprises an amino acid sequence (such as a CDR H3 region) selected fromSEQ ID NOS:52, 56, 58, 60, 62, and 64. In still other embodiments, theantibody further comprises an amino acid sequence shown in one or moreof SEQ ID NOS:18, 19, 30 and 31. In still other embodiments, theantibody further comprises one or more of the amino acid sequences shownin SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still other embodiments, theantibody further comprises one of more of the amino acid sequences shownin SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

In another aspect, the antibody may comprise:

(a) a heavy chain variable region comprising:

(i) a CDR1 region of SEQ ID NO: 30;

(ii) a CDR2 region comprising the sequence of SEQ ID NO: 31;

(iii) a CDR3 region selected from the group consisting of SEQ ID NO: 11,56, 58, 60, 62 and 64; and

(b) a light chain variable region comprising:

(i) a CDR1 region of SEQ ID NO: 18;

(ii) a CDR2 region of SEQ ID NO: 19;

(iii) a CDR3 region selected from the group consisting of SEQ ID NO: 14,55, 57, 59, 61 and 63.

In another aspect, the antibody comprises one or more of an amino acidsequence (such as a CDR region) shown in SEQ ID NOS:61, 63, 18, 19, 30and 31.

In another aspect, the antibody may be selected from an anti-NGFantibody known in the art, such as antibodies described in WO2005019266(including antibodies 4D4, 14D10, 6G9, 7H2, 14F11 and 4G6) orWO2006131951 (including antibody Hu-αD11). The antibody may bind to thesame epitope on NGF, particularly human NGF, as an anti-NGF antibodyknown in the art, such as antibodies described in WO2005019266(including antibodies 4D4, 14D10, 6G9, 7H2, 14F11 and 4G6) orWO2006131951 (including antibody Hu-αD11), or an antibody definedherein, and/or may compete for binding to NGF with such an antibody.

In some embodiments, the C-terminal lysine of the heavy chain of any ofthe anti-NGF antibodies described herein is cleaved. In various cases,the heavy and light chains of the anti-NGF antibodies may optionallyinclude a signal sequence.

In one aspect, the antibody is an anti-NGF antagonist antibody thatbinds NGF (such as human NGF) with a high affinity. In some embodiments,high affinity is (a) binding NGF with a K_(D) of less than about 2 nM(such as any of about 1 nM, 800 pM, 600 pM, 400 pM, 200 pM, 100 pM, 90pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM 5 pM orless), and/or a k_(off) of slower than about 6×10⁻⁵ s⁻¹); and/or (b)inhibiting (reducing, and/or blocking) human NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15pM of NGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM,20 pM, 10 pM, or less; and/or (c) inhibiting (reducing, and/or blocking)human NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 (in the presence of about 1.5 pM of NGF) of about any of 50 pM, 40pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, or less; and/or (d)inhibiting (reducing, and/or blocking) rat NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15pM of NGF) of about any of 150 pM, 125 pM, 100 pM, 80 pM, 60 pM, 40 pM,30 pM, 20 pM, 10 pM, 5 pM, or less; and/or (e) inhibiting (reducing,and/or blocking) rat NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 (in the presence of about 1.5 pM of NGF) of aboutany of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM,or less; and/or (f) and/or bind NGF with higher affinity than does thetrkA receptor.

In another aspect, the antibodies (a) bind NGF (such as human NGF) witha K_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM, 600pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30pM, 20 pM, 10 pM, 5 pM or less), and/or a k_(off) of slower than about6×10⁻⁵ s⁻¹); and/or (b) inhibit human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 15 pM ofNGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM,10 pM, or less; and/or (c) inhibit human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 1.5 pMof NGF) of about any of 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM,2 pM, 1 pM, or less; and/or bind NGF with higher affinity than does thetrkA receptor. In some embodiments, the antibodies (a) bind NGF with aK_(D) of less than about 2 nM; and/or (b) inhibit human NGF-dependentsurvival of mouse E13.5 trigeminal neurons with an IC50 of about 100 pMor less, wherein the IC50 is measured in the presence of about 15 pMNGF; and/or (c) inhibit human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 of about 10 pM or less, wherein the IC50is measured in the presence of about 1.5 pM of NGF, wherein the IC50 ismeasured in the presence of about 15 pM NGF. In some embodiments, theantibodies (a) bind NGF with a K_(D) of less than about 100 pM; and/or(b) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 20 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM NGF; and/or (c) inhibit humanNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 2 pM or less, wherein the IC50 is measured in the presence ofabout 1.5 pM of NGF.

In some embodiments, the above antibodies are isolated. In someembodiments, the antibody is substantially purified. In still otherembodiments, the antibody is affinity matured. In some embodiments, theantibody comprises human framework sequences. In still otherembodiments, the antibody comprises one or more non-human frameworkresidues. In some embodiments, the antibody binds NGF (such as humanNGF) with a K_(D) of 2 nM or less. In some embodiments, the antibodycomprises one or more (such as 2, 3, 4, 5, 6, 7, 8, or more) human aminoacid substitutions relative to a non-human amino acid sequence (such asa variable region sequence, such as a CDR sequence, such as a frameworksequence). In some embodiments, the antibody comprises at least 1, atleast 2, or more such as at least 3, 4, 5, 6, or more amino acidsubstitutions relative to a parent polypeptide amino acid sequence (suchas an antibody 911 amino acid sequence, such as any one or more of SEQID NOs 9-14). In some embodiments, the binding affinity of the antibodyhas been altered (in some embodiments, increased) relative to a parentantibody (such as Mab 911) affinity. In still other embodiments, thebinding affinity of the antibody is lower than the binding affinity oftrkA receptor for NGF (such as human NGF). In some embodiments, theantibodies are human antibodies. In other embodiments, the antibodiesare humanized antibodies. In still other embodiments, the antibodies aremonoclonal antibodies. In some embodiments, the antibody is an affinitymatured antibody.

The invention utilises polynucleotides (including isolatedpolynucleotide) comprising polynucleotides encoding any of theantibodies of the embodiments above.

In another aspect, the invention provides an isolated polynucleotidecomprising a polynucleotide encoding a fragment or a region of theantibody E3 (interchangeably termed “E3” herein). In one embodiment, thefragment is a light chain of the antibody E3 as shown in FIG. 1B ofWO2004/058184. In another embodiment, the fragment is a heavy chain ofthe antibody E3 as shown in FIG. 1A of WO2004/058184. In yet anotherembodiment, the fragment contains one or more variable regions from alight chain and/or a heavy chain of the antibody E3. In yet anotherembodiment, the fragment contains one or more complementaritydetermining regions (CDRs) from a light chain and/or a heavy chain ofthe antibody E3 as shown in FIGS. 1A and 1B of WO2004/058184.

In another aspect, the invention is an isolated polynucleotidecomprising a polynucleotide that encodes for antibody E3. In someembodiments, the polynucleotide comprises either or both of thepolynucleotides shown in FIGS. 2 and 3 of WO2004/058184.

In another aspect, the invention utilises an isolated polynucleotidethat encodes for an E3 light chain with a deposit number of ATCC No.PTA-4893 or ATCC No. PTA-4894. In another aspect, the invention is anisolated polynucleotide that encodes for an E3 heavy chain with adeposit number of ATCC No. PTA-4895. In yet another aspect, the isolatedpolynucleotide comprises (a) a variable region encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894and (b) a variable region encoded in the polynucleotide with a depositnumber of ATCC No. PTA-4895. In another aspect, the isolatedpolynucleotide comprises (a) one or more CDR encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894;and/or (b) one or more CDR encoded in the polynucleotide with a depositnumber of ATCC No. PTA-4895.

In another aspect, the invention utilises polynucleotides encoding anyof the antibodies (including antibody fragments) or polypeptidesdescribed herein.

In another aspect, the invention utilises vectors (including expressionand cloning vectors) and host cells comprising any of thepolynucleotides disclosed herein.

In another aspect, the invention utilises a host cell comprising apolynucleotide encoding E3 light chain and a polynucleotide encoding E3heavy chain, wherein the polynucleotide(s) encoding E3 light chain has adeposit number of ATCC No. PTA-4893 and/or ATCC No. PTA-4894, and thepolynucleotide encoding E3 heavy chain has a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises polynucleotidecomprising (a) a variable region encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894 and/or (b) a variableregion encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises a polynucleotideencoding (a) one or more CDR encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894; and/or (b) one or moreCDR encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell is a mammalian cell.

In another aspect, the invention provides an anti-NGF antagonistantibody for use in the treatment or prevention of pain and/or a lowerurinary tract symptom (LUTS) associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome. Theanti-NGF antagonist antibody may be as described herein.

Another aspect of the invention provides the use of an anti-NGFantagonist antibody in the manufacture of a medicament for the treatmentor prevention of pain and/or a lower urinary tract symptom (LUTS)associated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome. The anti-NGF antagonist antibody may be asdescribed herein.

In another aspect, the invention is a pharmaceutical composition for usein the treatment or prevention of pain and/or a lower urinary tractsymptom (LUTS) associated with interstitial cystitis and/or painfulbladder syndrome and/or bladder pain syndrome, comprising any of thepolypeptides (including antibodies such as antibody E3), polynucleotidesor vectors described herein, such as pharmaceutical compositionscomprising the antibody E3 or a fragment of the antibody E3, and apharmaceutically acceptable excipient.

In another aspect, the invention provides kits and compositionscomprising any one or more of the compositions described herein. Thesekits, generally in suitable packaging and provided with appropriateinstructions, are useful for any of the methods described herein.

The invention also provides any of the compositions and kits describedfor any use described herein whether in the context of use as medicamentand/or use for manufacture of a medicament.

Preferred features of each aspect of the invention apply equally to eachother aspect mutatis mutandis.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides methods for treating orpreventing pain and/or a lower urinary tract symptom (LUTS) associatedwith interstitial cystitis and/or painful bladder syndrome and/orbladder pain syndrome in an individual by administration of atherapeutically effective amount of an anti-NGF antagonist antibody.

The invention disclosed herein also provides an anti-NGF antagonistantibody for use in the treatment or prevention of pain and/or a lowerurinary tract symptom (LUTS) associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome. Furtherprovided is the use of an anti-NGF antagonist antibody in themanufacture of a medicament for the treatment or prevention of painand/or a lower urinary tract symptom (LUTS) associated with interstitialcystitis and/or painful bladder syndrome and/or bladder pain syndrome.

The invention disclosed herein provides the use of anti-NGF antagonistantibodies that bind NGF (such as human NGF) with high affinity. In someembodiments, the invention utilises a humanized antibody, E3, whichbinds to NGF. The invention also utilises E3 polypeptides (includingantibodies) that bind NGF, and polynucleotides encoding E3 antibodyand/or polypeptide. The invention further provides the use of antibodiesand polypeptides derived from E3 that bind NGF.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B.Lippincott Company, 1993).

Definitions

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv, dAb), singlechain antibodies (ScFv), mutants thereof, chimeric antibodies,diabodies, fusion proteins comprising an antibody portion, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site. An antibody includes an antibody of any class,such as IgG, IgA, or IgM (or sub-class thereof), and the antibody neednot be of any particular class. Depending on the antibody amino acidsequence of the constant domain of its heavy chains, immunoglobulins canbe assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond tothe different classes of immunoglobulins are called alpha, delta,epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Chothia et al. (1989) Nature342:877; Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As usedherein, a CDR may refer to CDRs defined by either approach or by acombination of both approaches.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination. “Fv” is an antibody fragment thatcontains a complete antigen-recognition and -binding site. In atwo-chain Fv species, this region consists of a dimer of one heavy andone light chain variable domain in tight, non-covalent association. In asingle-chain Fv species, one heavy and one light chain variable domaincan be covalently linked by a flexible peptide linker such that thelight and heavy chains can associate in a dimeric structure analogous tothat in a two-chain Fv species. It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingspecificity on the surface of the VH-VL dimer. However, even a singlevariable domain (or half of a Fv comprising only 3 CDRs specific for anantigen) has the ability to recognize and bind antigen, althoughgenerally at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge regions. A F(ab)2 fragment is abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region.

A single chain antibody (scFc) is an antibody in which VL and VH regionsare paired to form a monovalent molecule via a synthetic linker thatenables them to be made as a single protein chain (Bird et al Science,242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883 (1988)).

Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites.

An antibody can have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody(diabody) has two different binding sites.

An “isolated antibody” is an antibody that (1) is not associated withnaturally-associated components, including other naturally-associatedantibodies, that accompany it in its native state, (2) is free of otherproteins from the same species, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an antigen. A population of monoclonal antibodiesis highly specific, being directed against a single antigenic site. Theterm “monoclonal antibody” encompasses not only intact monoclonalantibodies and full-length monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity and the ability tobind to an antigen. It is not intended to be limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.).

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orhas been made using any of the techniques for making human antibodiesknown in the art or disclosed herein. This definition of a humanantibody includes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide. One suchexample is an antibody comprising murine light chain and human heavychain polypeptides. Human antibodies can be produced using varioustechniques known in the art. In one embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314;Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter,1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,222:581). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.Alternatively, the human antibody may be prepared by immortalizing humanB lymphocytes that produce an antibody directed against a target antigen(such B lymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor; BCR),etc. Such effector functions generally require the Fc region to becombined with a binding domain (e.g. an antibody variable domain) andcan be assessed using various assays known in the art for evaluatingsuch antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

As used herein “antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

As used herein, “Fc receptor” and “FcR” describe a receptor that bindsto the Fc region of an antibody. The preferred FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one which binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

As used herein, the terms “E3”, “3E”, and “antibody E3” are usedinterchangeably to refer to an antibody comprising the amino acidsequence of the heavy chain and light chain variable regions shown inSEQ ID NO:1 and SEQ ID NO:2 (FIGS. 1A and 1B of WO2004/058184),respectively. The CDR portions of antibody E3 (including Chothia andKabat CDRs) are diagrammatically depicted in FIGS. 1A and 1B ofWO2004/058184. FIGS. 2 and 3 of WO2004/058184 show polynucleotidesencoding heavy and light chains, respectively, comprising the heavy andlight chain variable regions shown in FIGS. 1A and 1B, respectively. Thegeneration and characterization of E3 is described in the Examples ofWO2004/058184, the entire content of which is herein incorporated byreference. Different biological functions are associated with E3,including, but not limited to, ability to bind to NGF and inhibit NGFbiological activity and/or downstream pathway(s) mediated by NGFsignaling; and ability to inhibit NGF-dependent survival of mouse E13.5trigeminal neurons. As discussed herein, antibodies for use in theinvention may have any one or more of these characteristics. In someembodiments, the term “E3” refers to immunoglobulin encoded by (a) apolynucleotide encoding E3 light chain that has a deposit number of ATCCNo. PTA-4893 or ATCC No. PTA-4894, and (b) a polynucleotide encoding E3heavy chain that has a deposit number of ATCC No. PTA-4895.

As used herein, “immunospecific” binding of antibodies refers to theantigen specific binding interaction that occurs between theantigen-combining site of an antibody and the specific antigenrecognized by that antibody (i.e., the antibody reacts with the proteinin an ELISA or other immunoassay, and does not react detectably withunrelated proteins).

An epitope that “specifically binds”, or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an NGF epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other NGF epitopes or non-NGFepitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

The term “identity” refers to the percent “identity” of two amino acidsequences or of two nucleic acid sequences. The percent identity isgenerally determined by aligning the sequences for optimal comparisonpurposes (e.g. gaps can be introduced in the first sequence for bestalignment with the second sequence) and comparing the amino acidresidues or nucleotides at corresponding positions. The “best alignment”is an alignment of two sequences that results in the highest percentidentity. The percent identity is determined by comparing the number ofidentical amino acid residues or nucleotides within the sequences (i.e.,% identity=number of identical positions/total number of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucliec Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search that detects distant relationshipsbetween molecules (Id.) When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the GCG sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

As used herein, the term “nerve growth factor” and “NGF” refers to nervegrowth factor and variants thereof that retain at least part of thebiological activity of NGF. As used herein, NGF includes all mammalianspecies of native sequence NGF, including human, canine, feline, equine,or bovine.

“NGF receptor” refers to a polypeptide that is bound by or activated byNGF. NGF receptors include the TrkA receptor and the p75 receptor of anymammalian species, including, but are not limited to, human, canine,feline, equine, primate, or bovine.

As used herein, an “anti-NGF antagonist antibody” (interchangeablytermed “anti-NGF antibody”) refers to an antibody which is able to bindto NGF and inhibit NGF biological activity and/or downstream pathway(s)mediated by NGF signaling. An anti-NGF antagonist antibody encompassesantibodies that block, antagonize, suppress or reduce (includingsignificantly) NGF biological activity, including downstream pathwaysmediated by NGF signaling, such as receptor binding and/or elicitationof a cellular response to NGF. For purpose of the present invention, itwill be explicitly understood that the term “anti-NGF antagonistantibody” encompass all the previously identified terms, titles, andfunctional states and characteristics whereby the NGF itself, an NGFbiological activity (including but not limited to its ability to abilityto mediate any aspect of post-surgical pain), or the consequences of thebiological activity, are substantially nullified, decreased, orneutralized in any meaningful degree. In some embodiments, an anti-NGFantagonist antibody binds NGF and prevent NGF dimerization and/orbinding to an NGF receptor (such as p75 and/or trkA). In otherembodiments, an anti-NGF antibody binds NGF and prevents trkA receptordimerization and/or trkA autophosphorylation. Examples of anti-NGFantagonist antibodies are provided herein.

“Biological activity” of NGF generally refers to the ability to bind NGFreceptors and/or activate NGF receptor signaling pathways. Withoutlimitation, a biological activity includes any one or more of thefollowing: the ability to bind an NGF receptor (such as p75 and/ortrkA); the ability to promote trkA receptor dimerization and/orautophosphorylation; the ability to activate an NGF receptor signalingpathway; the ability to promote cell differentiation, proliferation,survival, growth and other changes in cell physiology, including (in thecase of neurons, including peripheral and central neuron) change inneuronal morphology, synaptogenesis, synaptic function, neurotransmitterand/or neuropeptide release and regeneration following damage; theability to promote survival of mouse E13.5 trigeminal neurons; and theability to mediate pain and/or lower urinary tract symptoms associatedwith interstitial cystitis and/or painful bladder syndrome and/orbladder pain syndrome.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement or alleviation of any aspect of pain and/or alower urinary tract symptom associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, one or more of the following: including lesseningseverity, alleviation of pain and/or a lower urinary tract symptomassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome including any aspect of pain (such asshortening duration of pain, reduction of pain sensitivity orsensation).

An “effective amount” of drug, compound, or pharmaceutical compositionis an amount sufficient to effect beneficial or desired resultsincluding clinical results such as alleviation or reduction in painsensation. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient totreat, ameliorate, reduce the intensity of and/or prevent pain or alower urinary tract symptom associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome. In someembodiments, the “effective amount” may reduce pain at rest (restingpain) or mechanically-induced pain (including pain following movement),or both, and it may be administered before, during or after painfulstimulus. As is understood in the clinical context, an effective amountof a drug, compound, or pharmaceutical composition may or may not beachieved in conjunction with another drug, compound, or pharmaceuticalcomposition. Thus, an “effective amount” may be considered in thecontext of administering one or more therapeutic agents, and a singleagent may be considered to be given in an effective amount if, inconjunction with one or more other agents, a desirable result may be oris achieved.

“Reducing incidence” of pain and/or a lower urinary tract symptomassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome means any of reducing severity (which caninclude reducing need for and/or amount of (e.g., exposure to) otherdrugs and/or therapies generally used for this conditions, including,for example, opiates), duration, and/or frequency (including, forexample, delaying or increasing time pain in an individual). As isunderstood by those skilled in the art, individuals may vary in terms oftheir response to treatment, and, as such, for example, a “method ofreducing incidence of pain and/or a lower urinary tract symptomassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome in an individual” reflects administeringthe anti-NGF antagonist antibody based on a reasonable expectation thatsuch administration may likely cause such a reduction in incidence inthat particular individual.

“Ameliorating” a pain and/or a lower urinary tract symptoms associatedwith interstitial cystitis and/or painful bladder syndrome and/orbladder pain syndrome means a lessening or improvement of one or moresymptoms of pain and/or a lower urinary tract symptoms associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome as compared to not administering an anti-NGF antagonistantibody. “Ameliorating” also includes shortening or reduction induration of a symptom.

“Palliating” pain and/or a lower urinary tract symptom associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome means lessening the extent of one or more undesirableclinical manifestations of pain and/or lower urinary tract symptoms inan individual or population of individuals.

As used therein, “delaying” the development of pain means to defer,hinder, slow, retard, stabilize, and/or postpone progression of painand/or a lower urinary tract symptom associated with interstitialcystitis and/or painful bladder syndrome and/or bladder pain syndrome.This delay can be of varying lengths of time, depending on the historyof the disease and/or individuals being treated. As is evident to oneskilled in the art, a sufficient or significant delay can, in effect,encompass prevention, in that the individual does not develop pain. Amethod that “delays” development of the symptom is a method that reducesprobability of developing the symptom in a given time frame and/orreduces extent of the symptoms in a given time frame, when compared tonot using the method. Such comparisons are typically based on clinicalstudies, using a statistically significant number of subjects.

“Pain” as used herein refers to pain of any etiology, including acuteand chronic pain, and any pain with an inflammatory component. As usedherein, “pain” includes nociception and the sensation of pain, and paincan be assessed objectively and subjectively, using pain scores andother methods well-known in the art. The pain can be primary orsecondary pain, as is well-known in the art.

“Pain associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome” as used herein refers to lowerabdominal (pelvic) pain; bladder pain; suprapubic pain; vaginal pain;pain in the penis, testicles, scrotum and perineum; urethral pain;dyspareneuria; pain, pressure or discomfort that may increase as thebladder fills.

“Lower urinary tract symptoms associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome” as usedherein refers to three groups of urinary symptoms, which may be definedas storage (irritative), voiding (obstructive) and post-micturitionsymptoms. Storage symptoms comprise urgency, frequency, nocturia,urgency incontinence and stress incontinence, which can be associatedwith overactive bladder (OAB) and benign prostatic hyperplasia (BPH).Voiding symptoms comprise hesitancy, poor flow, intermittency, strainingand dysuria. Post-micturition symptoms comprise terminal dribbling,post-void dribbling and a sense of incomplete emptying.

“Over Active Bladder (OAB)” is defined as urgency, with or without urgeincontinence, usually with frequency and nocturia [Abrams et al.,Neurourology and Urodynamics 21:167-178 (2002)]. Prevalence of OAB inmen and women is similar, with approximately 16% of the population ofthe USA suffering from the condition [Stewart et al, Prevalence ofOveractive Bladder in the United States: Results from the NOBLE Program;Abstract Presented at the 2^(nd) International Consultation onIncontinence, July 2001, Paris, France].

The terms OAB Wet and OAB Dry describe OAB patients with or withouturinary incontinence respectively. Previously, the cardinal symptom ofOAB was believed to be urinary incontinence. However, with the advent ofthe new terms this is clearly not meaningful for the large number ofsufferers who are not incontinent (i.e. OAB Dry patients). Thus, a 2001study from Liberman et al [‘Health Related Quality of Life Among Adultswith Symptoms of Overactive Bladder: Results From A US Community-BasedSurvey’; Urology 57(6), 1044-1050, 2001] examined the impact of all OABsymptoms on the quality of life of a community-based sample of the USpopulation. This study demonstrated that individuals suffering from OABwithout any demonstrable loss of urine have an impaired quality of lifewhen compared with controls.

“BPH” is a chronically progressive disease that can lead tocomplications such as acute urinary retention, recurrent urinary tractinfections, bladder stones and renal dysfunction. The prevalence andaverage severity of LUTS associated with BPH in men increases with age.BPH leads to an increase in prostate volume, creating urethral andbladder outflow obstruction as well as secondary changes in bladderfunction. The effects of this are manifested by both storage(irritative) and voiding (obstructive) symptoms.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” or “subject” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, farmanimals (such as cows), sport animals, pets (such as cats, dogs andhorses), primates, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody-antigeninteraction. The term “K_(on)”, as used herein, is intended to refer tothe on-rate or association rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant of an antibody-antigen interaction, which isobtained from the ratio of K_(off) to K_(on) and is expressed as a molarconcentration (M). K_(D) values for antibodies can be determined usingmethods well established in the art. One method for determining theK_(D) of an antibody is by using surface plasmon resonance, typicallyusing a biosensor system such as a Biacore® system.

Methods of Using Anti-NGF Antagonist Antibody for Treating or PreventingPain and/or a Lower Urinary Tract Symptom Associated with InterstitialCystitis and/or Painful Bladder Syndrome and/or Bladder Pain Syndrome

The invention provides methods for treating or preventing pain and/or alower urinary tract symptom associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome in individualsincluding mammals, both human and non-human. Accordingly, in one aspect,the invention provides methods of treating or preventing pain and/or alower urinary tract symptom associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome in an individualcomprising administering an effective amount of an anti-NGF antagonistantibody. Suitable anti-NGF antagonist antibodies are described herein.

In another aspect, the invention provides methods for reducing incidenceof, ameliorating, suppressing, palliating, and/or delaying the onset,the development or the progression of pain and/or a lower urinary tractsymptom associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome in an individual. Thus, in someembodiments, the anti-NGF antagonist antibody is administered prior todevelopment of pain and/or a lower urinary tract symptom associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome in an individual having interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome.

An anti-NGF antagonist antibody may be administered to an individual viaany suitable route. Examples of different administration route aredescribed herein.

In some embodiments, the anti-NGF antagonist antibody is administeredonce every week, once every two weeks, once every three weeks, onceevery four weeks, once every five weeks, once every six weeks, onceevery seven weeks, once every eight weeks, once every nine weeks, onceevery ten weeks, once every fifteen weeks, once every twenty weeks, onceevery twenty five weeks, or once every twenty six weeks. In someembodiments, the anti-NGF antagonist antibody is administered once everymonth, once every two months, once every three months, once every fourmonths, once every five months, or once every six months.

Pain relief or relief from lower urinary tract symptoms may becharacterized by time course of relief. Accordingly, in someembodiments, relief is observed within about 24 hours afteradministration of an anti-NGF antagonist antibody. In other embodiments,relief is observed within about 36, 48, 60, 72 hours or 4 days afteradministration of the anti-NGF antagonist antibody. In some embodiments,frequency and/or intensity of pain and/or lower urinary tract symptom isdiminished, and/or quality of life of those suffering the disease isincreased. In some embodiments, pain relief is provided for duration ofat least about 7 days, at least about 14 days, at least about 21 days,at least about 28 days, at least about 35 days, at least about 42 days,at least about 49 days, at least about 56 days, at least about 63 days,at least about 70 days, at least about 77 days, at least about 84 days,at least about 180 days, or longer after a single dose of the anti-NGFantagonist antibody.

Pain relief or relief from lower urinary tract symptoms may becharacterized by a change in a pain numerical rating scale (NRS), achange in the O'Leary-Sant Interstitial Cystitis Symptom Index (ICSI), achange in the O'Leary-Sant Interstitial Cystitis Problem Index (ICPI), achange in the Pelvic Pain and Urgency/Frequency (PUF) symptom scoreand/or changes in micturition variables including micturition frequency,nocturnal frequency, incontinence episode frequency, mean volume voidedper micturition, mean interstitial cystitis pain severity, urinaryurgency episodes, average sleep disturbance score, average painassociated with sexual activity pain score, global response assessment,patent reporting treatment impact assessment, treatment failure,biomarkers, safety endpoints and/or pharmacokinetic measures. Biomarkersmay include NGF, glycoprotein-51 (GP-51), antiproliferative factor (APF)and HB-Epidermal growth factor (HB-EGF), amongst others.

Exemplary anti-NGF antibodies for use in the methods of the invention(pertaining to pain and/or lower urinary tract symptoms associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome) are anti-NGF antagonist antibodies, which refer to anyantibody that blocks, suppresses or reduces (including significantly)NGF biological activity, including downstream pathways mediated by NGFsignaling, such as receptor binding and/or elicitation of a cellularresponse to NGF. The term “antagonist” implies no specific mechanism ofbiological action whatsoever, and is deemed to expressly include andencompass all possible pharmacological, physiological, and biochemicalinteractions with NGF and its consequences which can be achieved by avariety of different, and chemically divergent, compositions. Forpurpose of the present invention, it will be explicitly understood thatthe term “antagonist” encompasses all the previously identified terms,titles, and functional states and characteristics whereby the NGFitself, an NGF biological activity (including but not limited to itsability to mediate any aspect of pain), or the consequences of thebiological activity, are substantially nullified, decreased, orneutralized in any meaningful degree. In some embodiments, an anti-NGFantagonist antibody binds (physically interact with) NGF, binds to anNGF receptor (such as TrkA receptor and/or p75 receptor), and/or reduces(impedes and/or blocks) downstream NGF receptor signaling. In someembodiment, the anti-NGF antagonist antibody binds NGF (such as hNGF)and does not significantly bind to related neurotrophins, such as NT-3,NT4/5, and/or BDNF. In some embodiments, the anti-NGF antagonistantibody is not associated with an adverse immune response. In stillother embodiments, the anti-NGF antibody is humanized (such as antibodyE3 described herein). In some embodiments, the anti-NGF antibody isantibody E3 (as described herein). In other embodiments, the anti-NGFantibody comprises one or more CDR(s) of antibody E3 (such as one, two,three, four, five, or, in some embodiments, all six CDRs from E3). Inother embodiments, the antibody is human. In still other embodiments,the anti-NGF antibody comprises the amino acid sequence of the heavychain variable region shown in SEQ ID No. 1 (FIG. 1A of WO2004/058184)and/or the amino acid sequence of the light chain variable region shownin SEQ ID NO:2 (FIG. 1B of WO2004/058184). In still other embodiments,the antibody comprises a modified constant region, such as a constantregion that is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). In other embodiments, the constant region ismodified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCTWO9958752.

The antibodies for use in the methods of the invention are characterizedby any (one or more) of the following characteristics: (a) ability tobind to NGF; (b) ability to reduce and/or inhibit NGF biologicalactivity and/or downstream pathway(s) mediated by NGF signaling; (c)ability to reduce and/or inhibit NGF-dependent survival of mouse E13.5trigeminal neurons; (d) absence of any significant cross-reactivity toNT3, NT4/5, and/or BDNF; (e) ability to treat and/or prevent pain and/orlower urinary tract symptoms associated with interstitial cystitisand/or painful bladder syndrome and/or bladder pain syndrome; (f)ability to increase clearance of NGF; (g) ability to reduce or inhibitactivation of trkA receptor, as detected, for example, using kinasereceptor activation assay (KIRA) (see U.S. Pat. No. 6,027,927).

For purposes of this invention, the antibody reacts with NGF in a mannerthat inhibits NGF and/or downstream pathways mediated by the NGFsignaling function. In some embodiments, the anti-NGF antagonistantibody recognizes human NGF. In yet other embodiments, the anti-NGFantagonist antibody specifically binds human NGF. In some embodiment,the anti-NGF antagonist antibody does not significantly bind to relatedneurotrophins, such as NT-3, NT4/5, and/or BDNF. In still otherembodiments, the anti-NGF antibody is capable of binding NGF andeffectively inhibiting the binding of NGF to its TrkA and/or p75receptor in vivo and/or effectively inhibiting NGF from activating itsTrkA and/or p75 receptor. In still other embodiments, the anti-NGFantagonist antibody is a monoclonal antibody. In still otherembodiments, the anti-NGF antibody is humanized (such as antibody E3described herein). In some embodiments, the anti-NGF antibody is human.See for example, WO 2005/019266 which describes antibodies 4D4, 14D10,6G9, 7H2, 14F11 and 4G6. In one embodiment, the antibody is a humanantibody which recognizes one or more epitopes on human NGF. In anotherembodiment, the antibody is a mouse or rat antibody which recognizes oneor more epitopes on human NGF. In another embodiment, the antibodyrecognizes one or more epitopes on an NGF selected from the groupconsisting of: primate, canine, feline, equine, and bovine. In stillfurther embodiments, the anti-NGF antagonist antibody binds essentiallythe same NGF epitope as an antibody selected from any one or more of thefollowing: MAb 911, MAb 912 and MAb 938 (See Hongo, et al., Hybridoma19:215-227 (2000)); an antibody as defined herein (such as antibody E3);and/or an antibody described in WO20050019266 (including antibodies 4D4,14D10, 6G9, 7H2, 14F11 and 4G6) or WO2006131951 (including antibodyHu-αD11). In other embodiments, the antibody binds the same epitope asMab 911. In another embodiment, the antibody comprises a constant regionthat is immunologically inert (e.g., does not trigger complementmediated lysis or antibody dependent cell mediated cytotoxicity (ADCC)).ADCC activity can be assessed using methods disclosed in U.S. Pat. No.5,500,362. In some embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613-2624; PCT WO9958752.

In some embodiments, the anti-NGF antagonist antibody is a humanizedmouse anti-NGF monoclonal antibody termed antibody “E3”, any of the E3related antibodies described herein, or any fragments thereof.

The binding properties of antibody E3, which binds human NGF with highaffinity and slow dissociation kinetics, compared with parent murineanti-NGF monoclonal antibody 911, are summarized below. E3 binds humanNGF with an approximately 50-fold higher binding affinity than parentmouse antibody 911.

antibody k_(D) K_(off) K_(on) 911 (Fab)  3.7 nM   9 × 10⁻⁵ s⁻¹ 2.2 × 10⁴M⁻¹s⁻¹ E3 (Fab) 0.07 nM <4 × 10⁻⁵ s⁻¹   6 × 10⁵ M⁻¹s⁻¹

The E3 antibody and related antibodies also exhibit a strong capacity toantagonize human NGF, as assessed by in vitro assays described inExamples 2 and 3 of WO2004/058184 the content of which is incorporatedherein by reference. For example, antibody E3 antagonizes theNGF-dependent survival of mouse E13 trigeminal neurons at an IC50 ofabout 21 pM in the presence of 15 pM of human NGF, and about 1.2 pM inthe presence of 1.5 pM of human NGF.

Accordingly, in another aspect, the antibodies and polypeptides of theinvention may be further identified and characterized by: (h) highaffinity binding to human NGF with low dissociation kinetics (in someembodiments, with a K_(D) of less than about 2 nM, and/or a koff ofslower than about 6×10-5 s-1) and/or (i) ability to inhibit (block)NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 100 pM or less at about 15 pM of NGF (in some embodiments, humanNGF) and/or an IC50 of about 20 pM or less at about 1.5 pM of NGF.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, humanantibodies, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity, including glycosylation variants of antibodies, amino acidsequence variants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies).

In some embodiments, the invention utilises an antibody comprising alight chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4893 or ATCC No.PTA-4894. In another aspect, the antibody comprises a heavy chain thatis encoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4895. The present invention also utilisesvarious formulations of E3 and equivalent antibody fragments (e.g., Fab,Fab′, F(ab′)₂, Fv, Fc, etc.), single chain (ScFv), mutants thereof,fusion proteins comprising an antibody portion, and any other modifiedconfiguration of E3 that comprises an antigen (NGF) recognition site ofthe required specificity. The equivalent antibodies of E3, includingantibody and polypeptide fragments (which may or may not be antibodies)of E3, and polypeptides comprising polypeptide fragments of E3 areidentified and characterized by any (one or more) of the criteriadescribed above.

Accordingly, the invention utilises any of the following, orcompositions (including pharmaceutical compositions) comprising any ofthe following: (a) antibody E3; (b) a fragment or a region of theantibody E3; (c) a light chain of the antibody E3 as shown in SEQ ID No.17 (FIG. 1B of WO2004/058184); (c) a heavy chain of the antibody E3 asshown in SEQ ID No. 16 (FIG. 1A of WO2004/058184); (d) one or morevariable region(s) from a light chain and/or a heavy chain of theantibody E3; (e) one or more CDR(s) (one, two, three, four, five or sixCDRs) of antibody E3 shown in SEQ ID Nos. 3-8 (FIGS. 1A and 1B ofWO2004/058184); (f) CDR H3 from the heavy chain of antibody E3 shown inSEQ ID No. 5 (FIG. 1A of WO2004/058184); (g) CDR L3 from the light chainof antibody E3 shown in SEQ ID No. 8 (FIG. 1B of WO2004/058184); (h)three CDRs from the light chain of antibody E3 shown in SEQ ID Nos. 6-8(FIG. 1B of WO2004/058184); (i) three CDRs from the heavy chain ofantibody E3 shown in SEQ ID No. 3-5 (FIG. 1A of WO2004/058184); (j)three CDRs from the light chain and three CDRs from the heavy chain, ofantibody E3 shown in SEQ ID Nos 3-8 (FIGS. 1A and 1B of WO2004/058184);and (k) an antibody comprising any one of (b) through (j).

The CDR portions of antibody E3 (including Chothia and Kabat CDRs) arediagrammatically depicted in FIGS. 1A and 1B of WO2004/058184), andconsist of the following amino acid sequences:

(SEQ ID NO: 3) (a) heavy chain CDR 1 (“CDR H1”) GFSLIGYDLN;(SEQ ID NO: 4) (b) heavy chain CDR 2 (“CDR H2”) IIWGDGTTDYNSAVKS;(SEQ ID NO: 5) (c) heavy chain CDR 3 (“CDR H3”) GGYWYATSYYFDY;(SEQ ID NO: 6) (d) light chain CDR 1 (“CDR L1”) RASQSISNNLN;(SEQ ID NO: 7) (e) light chain CDR 2 (“CDR L2”) YTSRFHS; and(SEQ ID NO: 8) (f) light chain CDR 3 (“CDR L3”) QQEHTLPYT.

Determination of CDR regions is well within the skill of the art. It isunderstood that in some embodiments, CDRs can be a combination of theKabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”).In some embodiments, the CDRs comprise the Kabat CDR. In otherembodiments, the CDRs are the Chothia CDR.

In some embodiments, the antibody comprises at least one CDR that issubstantially homologous to at least one CDR, at least two, at leastthree, at least four, at least 5 CDRs of E3 (or, in some embodimentssubstantially homologous to all 6 CDRs of E3, or derived from E3). Otherembodiments include antibodies which have at least two, three, four,five, or six CDR(s) that are substantially homologous to at least two,three, four, five or six CDRs of E3 or derived from E3. It is understoodthat, for purposes of this invention, binding specificity and/or overallactivity (which may be in terms of treating and/or preventing pain orinhibiting NGF-dependent survival of E13.5 mouse trigeminal neurons) isgenerally retained, although the extent of activity may vary compared toE3 (may be greater or lesser).

In some embodiments, the antibody may comprise an amino acid sequence ofE3 that has any of the following: at least 5 contiguous amino acids, atleast 8 contiguous amino acids, at least about 10 contiguous aminoacids, at least about 15 contiguous amino acids, at least about 20contiguous amino acids, at least about 25 contiguous amino acids, atleast about 30 contiguous amino acids of a sequence of E3, wherein atleast 3 of the amino acids are from a variable region of E3. Theextended CDR sequences of Mab 911 are shown in FIGS. 1A and 1B ofWO2004/058184, and in SEQ ID NOS:9-14 herein. In one embodiment, thevariable region is from a light chain of E3. In another embodiment, thevariable region is from a heavy chain of E3. In another embodiment, the5 (or more) contiguous amino acids are from a complementaritydetermining region (CDR) of E3 shown in SEQ ID Nos 3-8 (FIGS. 1A and 1Bof WO2004/058184).

In another embodiment, the antibody comprises an amino acid sequence ofE3 that has any of the following: at least 5 contiguous amino acids, atleast 8 contiguous amino acids, at least about 10 contiguous aminoacids, at least about 15 contiguous amino acids, at least about 20contiguous amino acids, at least about 25 contiguous amino acids, atleast about 30 contiguous amino acids of a sequence of E3, wherein theE3 sequence comprises any one or more of: amino acid residue L29 ofCDRH1, 150 of CDRH2, W101 of CDRH3, and/or A103 of CDRH3; and/or aminoacid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91 E of CDRL3and/or H92 of CDRL3.

As is evident, throughout this disclosure, a sequential amino acidnumbering scheme is used to refer to amino acid residues in the variableregions (that is, the amino acid residues in each variable region arenumbered in sequence). As is well known in the art, the Kabat and/orChothia numbering systems are useful when comparing two antibodies orpolypeptides, such as an E3 antibody and an E3 variant (or polypeptidesuspected of being an E3 variant). It is well understood in the art howto convert sequential numbering to Chothia and/or Kabat numbering, ifdesired, for example, for use in making comparisons between E3 andanother polypeptide. FIG. 23 of WO2004/058184 depicts the E3 variableregions numbered using sequential, Chothia and Kabat numbering. Inaddition, to facilitate comparison, generally it is understood thatframework residues generally, but not always, have approximately thesame number of residues. However, the CDRs may vary in size (i.e., it ispossible to have insertions and/or deletions of one or more amino acidresidues). When comparing an E3 antibody and a candidate E3 variant (forexample, in the case of a CDR region from a candidate sequence which islonger in the sequence in antibody E3 to which is aligned), one mayfollow the following steps (though other methods are known in the art).The candidate antibody sequence is aligned with E3 antibody heavy chainand light chain variable regions. Alignment may be done by hand, or bycomputer using commonly accepted computer programs. Alignment may befacilitated by using some amino acid residues which are common to mostFab sequences. For example, the light and heavy chains each typicallyhave two cysteines, which are often found at a conserved position. It isunderstood that the amino acid sequence of a candidate variant antibodymay be longer (i.e. have inserted amino acid residues) or shorter (havedeleted amino acid residues). Suffixes may be added to the residuenumber to indicate the insertion of additional residues, e.g., residue34 abc. For candidate sequences which, for example, align with a E3sequence for, e.g., residues 33 and 35, but have no residue between themto align with residue 35, the residue 35 is simply not assigned to aresidue. In another approach, it is generally well known that comparisonmay be made between structural equivalent (e.g., same position in theantigen-antibody complex) amino acids when comparing CDRs of differentlengths. For example, the Chothia numbering (Al-Lazikani et al, supra)generally (but not in all cases), places insertions and deletions at thestructurally correct positions. Structural equivalence may also bededuced or demonstrated using X-ray crystallography or double mutantcycle analysis (see Pons et al. (1999) Prot. Sci. 8:958-968).

The binding affinity of an anti-NGF antibody to NGF (such as hNGF) canbe, in terms of K_(D), about 0.10 to about 0.80 nM, about 0.15 to about0.75 nM and about 0.18 to about 0.72 nM. In some embodiments, the K_(D)is about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about40 pM, or greater than about 40 pM. In one embodiment, the K_(D) isbetween about 2 pM and 22 pM. In other embodiments, the K_(D) is lessthan about 10 nM, about 5 nM, about 4 nM, about 3.5 nM, about 3 nM,about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about300 pM, about 200 pM, about 150 pM, about 100 pM, about 90 pM, about 80pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM,about 10 pM, about 5 pM. In some embodiments, the K_(D) is about 10 nM.In other embodiments, the K_(D) is less than about 10 nM. In otherembodiments, the K_(D) is about 0.1 nM or about 0.07 nM. In otherembodiments, the K_(D) is less than about 0.1 nM or less than about 0.07nM. In other embodiments, the K_(D) is any of about 10 nM, about 5 nM,about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about1.5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about 600pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM,about 50 pM, about 40 pM, about 30 pM, about 10 pM to any of about 2 pM,about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM. Insome embodiments, the K_(D) is any of about 10 nM, about 5 nM, about 4nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM,about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM,about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM,about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about50 pM, about 40 pM, about 30 pM, about 10 pM. In still otherembodiments, the K_(D) is about 2 pM, about 5 pM, about 10 pM, about 15pM, about 20 pM, about 40 pM, or greater than about 40 pM.

The binding affinity of the antibody to NGF can be determined usingmethods well known in the art. One way of determining binding affinityof antibodies to NGF is by measuring binding affinity of monofunctionalFab fragments of the antibody. To obtain monofunctional Fab fragments,an antibody (for example, IgG) can be cleaved with papain or expressedrecombinantly. The affinity of an anti-NGF Fab fragment of an antibodycan be determined by surface plasmon resonance (BIAcore3000™ surfaceplasmon resonance (SPR) system, BIAcore, INC, Piscaway N.J.). CM5 chipscan be activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiinidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Human NGF (or any other NGF) can be dilutedinto 10 mM sodium acetate pH 4.0 and injected over the activated chip ata concentration of 0.005 mg/mL. Using variable flow time across theindividual chip channels, two ranges of antigen density can be achieved:100-200 response units (RU) for detailed kinetic studies and 500-600 RUfor screening assays. The chip can be blocked with ethanolamine.Regeneration studies have shown that a mixture of Pierce elution buffer(Product No. 21004, Pierce Biotechnology, Rockford Ill.) and 4 M NaCl(2:1) effectively removes the bound Fab while keeping the activity ofhNGF on the chip for over 200 injections. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15 NaCl, 3 mM EDTA, 0.005% Surfactant P29) is used as runningbuffer for the BIAcore assays. Serial dilutions (0.1-10× estimatedK_(D)) of purified Fab samples are injected for 1 min at 100 μL/min anddissociation times of up to 2 h are allowed. The concentrations of theFab proteins are determined by ELISA and/or SDS-PAGE electrophoresisusing a Fab of known concentration (as determined by amino acidanalysis) as a standard. Kinetic association rates (k_(on)) anddissociation rates (k_(off)) are obtained simultaneously by fitting thedata to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam,L. Petersson, B. (1994). Methods Enzymology 6.99-110) using theBIAevaluation program. Equilibrium dissociation constant (K_(D)) valuesare calculated as k_(off)/k_(on). This protocol is suitable for use indetermining binding affinity of an antibody to any NGF, including humanNGF, NGF of another vertebrate (in some embodiments, mammalian) (such asmouse NGF, rat NGF, primate NGF), as well as for use with otherneurotrophins, such as the related neurotrophins NT3, NT4/5, and/orBDNF.

In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiment, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

The epitope(s) can be continuous or discontinuous. In one embodiment,the antibody binds essentially the same human NGF epitopes as anantibody selected from the group consisting of MAb 911, MAb 912, and MAb938 as described in Hongo et al., Hybridoma, 19:215-227 (2000); anantibody defined herein (such as antibody E3); and/or an antibodydescribed in WO2005019266 (including antibodies 4D4, 14D10, 6G9, 7H2,14F11 and 4G6) or WO2006131951 (including antibody Hu-αD11), the entirecontents of which are herein incorporated by reference. In anotherembodiment, the antibody binds essentially the same hNGF epitope as MAb911. In still another embodiment, the antibody binds essentially thesame epitope as MAb 909. Hongo et al., supra. For example, the epitopemay comprise one or more of: residues K32, K34 and E35 within variableregion 1 (amino acids 23-35) of hNGF; residues F79 and T81 withinvariable region 4 (amino acids 81-88) of hNGF; residues H84 and K88within variable region 4; residue R103 between variable region 5 (aminoacids 94-98) of hNGF and the C-terminus (amino acids 111-118) of hNGF;residue E11 within pre-variable region 1 (amino acids 10-23) of hNGF;Y52 between variable region 2 (amino acids 40-49) of hNGF and variableregion 3 (amino acids 59-66) of hNGF; residues L112 and S113 within theC-terminus of hNGF; residues R59 and R69 within variable region 3 ofhNGF; or residues V18, V20, and G23 within pre-variable region 1 ofhNGF. In addition, an epitope can comprise one or more of the variableregion 1, variable region 3, variable region 4, variable region 5, theN-terminus region, and/or the C-terminus of hNGF. In still anotherembodiment, the antibody significantly reduces the solvent accessibilityof residue R103 of hNGF. It is understood that although the epitopesdescribed above relate to human NGF, one of ordinary skill can align thestructures of human NGF with the NGF of other species and identifylikely counterparts to these epitopes.

In some embodiments, the antibodies for use in the invention may inhibit(reduce, and/or block) human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF)of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10pM, or less. In some embodiments, the antibodies or peptides of theinvention may inhibit (reduce, and/or block) human NGF-dependentsurvival of mouse E13.5 trigeminal neurons with an IC50 (in the presenceof about 1.5 pM of NGF) of about any of 50 pM, 40 pM, 30 pM, 10 pM, 20pM, 10 pM, 5 pM, 2 pM, 1 pM, or less. In some embodiments, theantibodies or peptides of the invention may inhibit (reduce, and/orblock) rat NGF-dependent survival of mouse E13.5 trigeminal neurons withan IC50 (in the presence of about 15 pM of NGF) of about any of 150 pM,125 pM, 100 pM, 80 pM, 60 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or less.In some embodiments, the antibodies may inhibit (reduce, and/or block)rat NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 (in the presence of about 1.5 pM of NGF) of about any of 30 pM, 25pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, or less. Methodsfor measurement of the NGF-dependent survival of mouse E13 trigeminalneurons are known in the art, and described, e.g., in Example 2 ofWO2004/058184).

Identification of Anti-NGF Antagonist Antibodies

Anti-NGF antagonist antibodies for use in the invention can beidentified or characterized using methods known in the art, wherebyreduction, amelioration, or neutralization of an NGF biological activityis detected and/or measured. Methods described in PCT WO 04/065560 canbe used. Another method, for example, a kinase receptor activation(KIRA) assay described in U.S. Pat. Nos. 5,766,863 and 5,891,650, can beused to identify anti-NGF agents. This ELISA-type assay is suitable forqualitative or quantitative measurement of kinase activation bymeasuring the autophosphorylation of the kinase domain of a receptorprotein tyrosine kinase (hereinafter “rPTK”), e.g. TrkA receptor, aswell as for identification and characterization of potential antagonistsof a selected rPTK, e.g., TrkA. The first stage of the assay involvesphosphorylation of the kinase domain of a kinase receptor, for example,a TrkA receptor, wherein the receptor is present in the cell membrane ofan eukaryotic cell.

The receptor may be an endogenous receptor or nucleic acid encoding thereceptor, or a receptor construct, may be transformed into the cell.Typically, a first solid phase (e.g., a well of a first assay plate) iscoated with a substantially homogeneous population of such cells(usually a mammalian cell line) so that the cells adhere to the solidphase. Often, the cells are adherent and thereby adhere naturally to thefirst solid phase. If a “receptor construct” is used, it usuallycomprises a fusion of a kinase receptor and a flag polypeptide. The flagpolypeptide is recognized by the capture agent, often a captureantibody, in the ELISA part of the assay. An analyte, such as acandidate NGF antagonist (including anti-NGF antagonist antibody) isthen added together with NGF to the wells having the adherent cells,such that the tyrosine kinase receptor (e.g. TrkA receptor) is exposedto (or contacted with) NGF and the analyte. This assay enablesidentification of antagonists (including antibodies) that inhibitactivation of TrkA by its ligand NGF. Following exposure to NGF and theanalyte, the adhering cells are solubilized using a lysis buffer (whichhas a solubilizing detergent therein) and gentle agitation, therebyreleasing cell lysate which can be subjected to the ELISA part of theassay directly, without the need for concentration or clarification ofthe cell lysate.

The cell lysate thus prepared is then ready to be subjected to the ELISAstage of the assay. As a first step in the ELISA stage, a second solidphase (usually a well of an ELISA microtiter plate) is coated with acapture agent (often a capture antibody) which binds specifically to thetyrosine kinase receptor, or, in the case of a receptor construct, tothe flag polypeptide. Coating of the second solid phase is carried outso that the capture agent adheres to the second solid phase. The captureagent is generally a monoclonal antibody, but, as is described in theexamples herein, polyclonal antibodies may also be used. The cell lysateobtained is then exposed to, or contacted with, the adhering captureagent so that the receptor or receptor construct adheres to (or iscaptured in) the second solid phase. A washing step is then carried out,so as to remove unbound cell lysate, leaving the captured receptor orreceptor construct. The adhering or captured receptor or receptorconstruct is then exposed to, or contacted with, an anti-phosphotyrosineantibody which identifies phosphorylated tyrosine residues in thetyrosine kinase receptor. In one embodiment, the anti-phosphotyrosineantibody is conjugated (directly or indirectly) to an enzyme whichcatalyses a color change of a non-radioactive color reagent.Accordingly, phosphorylation of the receptor can be measured by asubsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

Anti-NGF antagonist antibodies can also be identified by incubating acandidate agent with NGF and monitoring any one or more of the followingcharacteristics: (a) binding to NGF and inhibiting NGF biologicalactivity or downstream pathways mediated by NGF signaling function; (b)inhibiting, blocking or decreasing NGF receptor activation (includingTrkA dimerization and/or autophosphorylation); (c) increasing clearanceof NGF; (d) treating or preventing any aspect of pain and/or lowerurinary tract symptoms associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome; (e) inhibiting(reducing) NGF synthesis, production or release. In some embodiments, anNGF antagonist (e.g., an anti-NGF antagonist antibody) is identified byincubating an candidate agent with NGF and monitoring binding and/orattendant reduction or neutralization of a biological activity of NGF.The binding assay may be performed with purified NGF polypeptide(s), orwith cells naturally expressing, or transfected to express, NGFpolypeptide(s). In one embodiment, the binding assay is a competitivebinding assay, where the ability of a candidate agent (such as anantibody) to compete with a known anti-NGF antagonist antibody for NGFbinding is evaluated. The assay may be performed in various formats,including the ELISA format. In other embodiments, an NGF antagonist(such as anti-NGF antagonist antibody) is identified by incubating acandidate agent with NGF and monitoring binding and attendant inhibitionof trkA receptor dimerization and/or autophosphorylation.

Following initial identification, the activity of a candidate anti-NGFantagonist antibody can be further confirmed and refined by bioassays,known to test the targeted biological activities. Alternatively,bioassays can be used to screen candidates directly. For example, NGFpromotes a number of morphologically recognizable changes in responsivecells. These include, but are not limited to, promoting thedifferentiation of PC12 cells and enhancing the growth of neurites fromthese cells (Greene et al., Proc Natl Acad Sci USA. 73(7):2424-8, 1976),promoting neurite outgrowth from explants of responsive sensory andsympathetic ganglia (Levi-Montalcini, R. and Angeletti, P. Nerve growthfactor. Physiol. Rev. 48:534-569, 1968) and promoting the survival ofNGF dependent neurons such as embryonic dorsal root ganglion, trigeminalganglion, or sympathetic ganglion neurons (e.g., Chun & Patterson, Dev.Biol. 75:705-711, (1977); Buchman & Davies, Development 118:989-1001(1993). Thus, the assay for inhibition of NGF biological activity entailculturing NGF responsive cells with NGF plus an analyte, such as acandidate NGF antagonist (including anti-NGF antagonist antibody). Afteran appropriate time the cell response will be assayed (celldifferentiation, neurite outgrowth or cell survival).

The ability of a candidate NGF antagonist antibody to block orneutralize a biological activity of NGF can also be assessed bymonitoring the ability of the candidate agent to inhibit NGF mediatedsurvival in the embryonic rat dorsal root ganglia survival bioassay asdescribed in Hongo et al., Hybridoma 19:215-227 (2000).

The antibodies for use in this invention can be made by procedures knownin the art, some of which are illustrated in the Examples ofWO2004/058184. The antibodies can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described in WO2004/058184 or by chemicalsynthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, a E3 antibody could beproduced by an automated polypeptide synthesizer employing the solidphase method. See also, U.S. Pat. Nos. 5,807,715; 4,816,567; and6,331,415. Chimeric or hybrid antibodies also may be prepared in vitrousing known methods of synthetic protein chemistry, including thoseinvolving cross-linking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

In another alternative, the antibodies can be made recombinantly usingprocedures that are well known in the art. In one embodiment, apolynucleotide comprising a sequence encoding the variable and lightchain regions of antibody E3 is cloned into a vector for expression orpropagation in a host cell (e.g., CHO cells). In another embodiment, thepolynucleotide sequences shown in FIGS. 2 and 3 of WO2004/058184 arecloned into one or more vectors for expression or propagation. Thesequence encoding the antibody of interest may be maintained in a vectorin a host cell and the host cell can then be expanded and frozen forfuture use. Vectors (including expression vectors) and host cells arefurther described herein. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters et al. (2001) Vaccine 19:2756; Lonberg, N. and D. Huszar (1995)Int. Rev. Immunol 13:65; and Pollock et al. (1999) J Immunol Methods231:147. Methods for making derivatives of antibodies, e.g., humanized,single chain, etc. are known in the art.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as E3. Single chainvariable region fragments are made by linking light and/or heavy chainvariable regions by using a short linking peptide. Bird et al. (1988)Science 242:423-426. An example of a linking peptide is (GGGGS)3 (SEQ IDNO:15), which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used (Bird etal. (1988)). Linkers can in turn be modified for additional functions,such as attachment of drugs or attachment to solid supports. The singlechain variants can be produced either recombinantly or synthetically.For synthetic production of scFv, an automated synthesizer can be used.For recombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

The antibody may be a bispecific antibody, a monoclonal antibody thathas binding specificities for at least two different antigens. Abispecific antibody can be prepared using the antibodies disclosedherein. Methods for making bispecific antibodies are known in the art(see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210).Traditionally, the recombinant production of bispecific antibodies wasbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, with the two heavy chains having different specificities(Millstein and Cuello, 1983, Nature 305, 537-539).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690, published Mar. 3, 1994.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods.

Suitable cross-linking agents and techniques are well known in the art,and are described in U.S. Pat. No. 4,676,980.

The antibody may be a humanized antibody, for example, as known in theart, and as described herein.

The invention encompasses modifications to antibody E3, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activity.Modification of polypeptides is routine practice in the art and isfurther exemplified in the Examples. Examples of modified polypeptidesinclude polypeptides with substitutions (including conservativesubstitutions) of amino acid residues, one or more deletions oradditions of amino acids which do not significantly deleteriously changethe functional activity, or use of chemical analogs.

A polypeptide “variant,” as used herein, is a polypeptide that differsfrom a native protein in one or more substitutions, deletions, additionsand/or insertions, such that the immunoreactivity of the polypeptide isnot substantially diminished. In other words, the ability of a variantto specifically bind antigen may be enhanced or unchanged, relative tothe native protein, or may be diminished by less than 50%, andpreferably less than 20%, relative to the native protein. Polypeptidevariants preferably exhibit at least about 80%, more preferably at leastabout 90% and most preferably at least about 95% identity (determined asdescribed herein) to the identified polypeptides.

Amino acid sequence variants of the antibodies may be prepared byintroducing appropriate nucleotide changes into the antibody DNA, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of SEQ ID NO:1 or 2 described herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis or modification iscalled “alanine scanning mutagenesis,” and is described by Cunninghamand Wells, 1989, Science, 244:1081-1085. A residue or group of targetresidues is identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity. Library scanningmutagenesis, as described herein, may also be used to identify locationsin an antibody that are suitable for mutagenesis or modification.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“conservative substitutions”. If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Amino Acid Substitutions Conservative Original ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp, Glu;

(4) Basic: Asn, Gln, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiment, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR3 domain. In still other embodiments,the CDR domain is CDRH3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GIcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H). In addition, the recombinant host cell canbe genetically engineered to be defective in processing certain types ofpolysaccharides. These and similar techniques are well known in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified E3 polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572, published Nov. 18,1999. These antibodies comprise, in addition to a binding domaindirected at the target molecule, an effector domain having an amino acidsequence substantially homologous to all or part of a constant domain ofa human immunoglobulin heavy chain. These antibodies are capable ofbinding the target molecule without triggering significant complementdependent lysis, or cell-mediated destruction of the target. In someembodiments, the effector domain is capable of specifically binding FcRnand/or FcγRIIb. These are typically based on chimeric domains derivedfrom two or more human immunoglobulin heavy chain CH2 domains.Antibodies modified in this manner are particularly suitable for use inchronic antibody therapy, to avoid inflammatory and other adversereactions to conventional antibody therapy.

The invention also utilises fusion proteins comprising one or morefragments or regions from the antibodies or polypeptides of thisinvention. In one embodiment, a fusion polypeptide is provided thatcomprises at least 10 contiguous amino acids of the variable light chainregion shown in SEQ ID No. 2 (FIG. 1B of WO2004/058184) and/or at least10 amino acids of the variable heavy chain region shown in SEQ ID No. 1(FIG. 1A of WO2004/058184). In another embodiment, the fusionpolypeptide comprises a light chain variable region and/or a heavy chainvariable region of E3, as shown in SEQ ID Nos. 1 and 2 (FIGS. 1A and 1Bof WO2004/058184). In another embodiment, the fusion polypeptidecomprises one or more CDR(s) of E3. In still other embodiments, thefusion polypeptide comprises CDR H3 and/or CDR L3 of antibody E3. Inanother embodiment, the fusion polypeptide comprises any one or more of:amino acid residue L29 of CDRH1, 150 of CDRH2, W101 of CDRH3, and/orA103 of CDRH3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51of CDRL2, 91 E of CDRL3 and/or H92 of CDRL3. For purposes of thisinvention, a E3 fusion protein contains one or more E3 antibodies andanother amino acid sequence to which it is not attached in the nativemolecule, for example, a heterologous sequence or a homologous sequencefrom another region. Exemplary heterologous sequences include, but arenot limited to a “tag” such as a FLAG tag or a 6His tag. Tags are wellknown in the art.

A E3 fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the E3 fusionproteins are made by preparing an expressing a polynucleotide encodingthem using recombinant methods described herein, although they may alsobe prepared by other means known in the art, including, for example,chemical synthesis.

The ability of the antibodies and polypeptides of this invention, suchas binding NGF; reducing or inhibiting a NGF biological activity;reducing and/or blocking NGF-induced survival of E13.5 mouse trigeminalneurons, may be tested using methods known in the art, some of which aredescribed in the Examples of WO2004/058184 and in particular in Examples2 and 3 of WO2004/058184.

The invention also utilises compositions (including pharmaceuticalcompositions) and kits comprising antibody E3, and, as this disclosuremakes clear, any or all of the antibodies and/or polypeptides describedherein. Such compositions and kits may be for use in treating and/orpreventing one or more pain and/or lower urinary tract symptomsassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome in an individual.

Polynucleotides, Vectors and Host Cells

The invention also provides isolated polynucleotides encoding theantibodies and polypeptides of the invention (including an antibodycomprising the polypeptide sequences of the light chain and heavy chainvariable regions shown in FIGS. 1A and 1B of WO2004058184), and vectorsand host cells comprising the polynucleotide for use in the methods ofthe invention.

Accordingly, the polynucleotides (or compositions, includingpharmaceutical compositions), may comprise polynucleotides encoding anyof the following: (a) antibody E3; (b) a fragment or a region of theantibody E3; (c) a light chain of the antibody E3 as shown in SEQ ID No.17 (FIG. 1B of WO2004/058184); (d) a heavy chain of the antibody E3 asshown in SEQ ID No. 16 (FIG. 1A of WO2004/058184); (e) one or morevariable region(s) from a light chain and/or a heavy chain of theantibody E3; (f) one or more CDR(s) (one, two, three, four, five or sixCDRs) of antibody E3 shown in SEQ ID Nos. 3-8 (FIGS. 1A and 1B ofWO2004/058184); (g) CDR H3 from the heavy chain of antibody E3 shown SEQID No. 5 (FIG. 1A of WO2004/058184; (h) CDR L3 from the light chain ofantibody E3 shown in SEQ ID No. 8 (FIG. 1B of WO2004/058184); (i) threeCDRs from the light chain of antibody E3 shown in SEQ ID No. 6-8 (FIG.1B of WO2004/058184); (j) three CDRs from the heavy chain of antibody E3shown in SEQ ID Nos. 3-5 (FIG. 1A of WO2004/058184); (k) three CDRs fromthe light chain and three CDRs from the heavy chain, of antibody E3shown in SEQ ID Nos. 3-8 (FIGS. 1A and 1B of WO2004/058184; or (I) anantibody comprising any of (b) to (k). In some embodiments, thepolynucleotide comprises either or both of the polynucleotide(s) shownin SEQ ID Nos. 76 and 77 (FIGS. 2 and 3 of WO2004/058184).

In another aspect, the invention utilises an isolated polynucleotidethat encodes for an E3 light chain with a deposit number of ATCC No.PTA-4893 or ATCC No. PTA-4894. In another aspect, the invention utilisesan isolated polynucleotide that encodes for an E3 heavy chain with adeposit number of ATCC No. PTA-4895. In yet another aspect, theinvention utilises an isolated polynucleotide comprising (a) a variableregion encoded in the polynucleotide with a deposit number of ATCC No.PTA-4894 and (b) a variable region encoded in the polynucleotide with adeposit number of ATCC No. PTA-4895. In another aspect, the inventionutilises an isolated polynucleotide comprising (a) one or more CDRencoded in the polynucleotide with a deposit number of ATCC No.PTA-4894; and/or (b) one or more CDR encoded in the polynucleotide witha deposit number of ATCC No. PTA-4895. The deposits under ATCC AccessionNos. PTA-4893, PTA-4894 and PTA-4895 are described in WO2004/058184, thecontent of which is incorporated herein in its entirety.

The polynucleotides encoding any of the antibodies (including antibodyfragments) and polypeptides described herein can be made by proceduresknown in the art

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention for use in treating or preventing pain and/or a lowerurinary tract symptom associated with interstitial cystitis and/orpainful bladder syndrome and/or bladder pain syndrome. In someembodiments, the composition comprises an expression vector comprising apolynucleotide encoding the E3 antibody as described herein. In otherembodiment, the composition comprises an expression vector comprising apolynucleotide encoding any of the antibodies or polypeptides describedherein. In still other embodiments, the composition comprises either orboth of the polynucleotides shown in FIGS. 2 and 3 of WO2004/058184.Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein for use in methods for treating orpreventing pain and/or a lower urinary tract symptom associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably at least about 80% identity and most preferably at leastabout 90% identity to a polynucleotide sequence that encodes a nativeantibody or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or10 to 12 percent, as compared to the reference sequences (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides for use in the invention can be obtained usingchemical synthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al. (1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., (1989), for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also utilises host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably 10 fold higher, even morepreferably 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to NGF is effected by animmunoassay or FACS. A cell overexpressing the antibody or protein ofinterest can be identified.

Compositions for Use in the Methods of the Invention

The compositions for use in the methods of the invention (pertaining topain and/or lower urinary tract symptoms associated with interstitialcystitis and/or painful bladder syndrome and/or bladder pain syndrome)comprise an effective amount of an anti-NGF antagonist antibody, and, insome embodiments, further comprise a pharmaceutically acceptableexcipient. Examples of such compositions, as well as how to formulate,are also described herein. In some embodiments, the anti-NGF antagonistantibody binds NGF and does not significantly cross-react with relatedneurotrophins (such as NT3, NT4/5, and/or BDNF). In some embodiments,the anti-NGF antagonist antibody is not associated with an adverseimmune response. In other embodiments, the anti-NGF antibody recognizeshuman NGF. In some embodiments, the anti-NGF antibody is human. In stillother embodiments, the anti-NGF antibody is humanized (such as antibodyE3 described herein). In still other embodiment, the anti-NGF antibodycomprises a constant region that does not trigger an unwanted orundesirable immune response, such as antibody-mediated lysis or ADCC. Inother embodiments, the anti-NGF antibody comprises one or more CDR(s) ofantibody E3 (such as one, two, three, four, five, or, in someembodiments, all six CDRs from E3).

It is understood that the compositions can comprise more than one NGFantagonist. For example, a composition can comprise more than one memberof a class of NGF antagonist (e.g., a mixture of anti-NGF antibodiesthat recognize different epitopes of NGF), as well as members ofdifferent classes of NGF antagonists (e.g., an anti-NGF antibody and anNGF inhibitory compound). Other exemplary compositions comprise morethan one anti-NGF antibodies that recognize the same epitope(s),different species of anti-NGF antibodies that bind to different epitopesof NGF, or different NGF inhibitory compounds.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and Practice of Pharmacy 20th Ed. (2000)Lippincott Williams and Wilkins, Ed. K. E. Hoover.), in the form oflyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable excipients are further described herein.

The anti-NGF antagonist antibody and compositions thereof can also beused in conjunction with other agents that serve to enhance and/orcomplement the effectiveness of the agents, for example with agents usedin the treatment or prevention of pain and/or lower urinary tractsymptoms. The anti-NGF antagonist antibody may be administeredsimultaneously, sequentially or separately in combination with one ormore of such agents.

The anti-NGF antibody may be administered in combination with one ormore other drugs (or as any combination thereof). The anti-NGF antibodymay be usefully combined with another pharmacologically active compound,or with two or more other pharmacologically active compounds, for thetreatment of a pain and/or a lower urinary tract symptom (LUTS)associated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome. For example, an anti-NGF antibody, asdefined above, may be administered simultaneously, sequentially orseparately, in combination with one or more agents selected from:

-   -   an opioid analgesic, e.g. morphine, heroin, hydromorphone,        oxymorphone, levorphanol, levallorphan, methadone, meperidine,        fentanyl, cocaine, codeine, dihydrocodeine, oxycodone,        hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone,        naltrexone, buprenorphine, butorphanol, nalbuphine or        pentazocine;    -   a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin,        diclofenac, diflusinal, etodolac, fenbufen, fenoprofen,        flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen,        ketorolac, meclofenamic acid, mefenamic acid, meloxicam,        nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine,        oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac,        tolmetin or zomepirac;    -   a barbiturate sedative, e.g. amobarbital, aprobarbital,        butabarbital, butabital, mephobarbital, metharbital,        methohexital, pentobarbital, phenobartital, secobarbital,        talbutal, theamylal or thiopental;    -   a benzodiazepine having a sedative action, e.g.        chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,        oxazepam, temazepam or triazolam;    -   an H₁ antagonist having a sedative action, e.g. diphenhydramine,        pyrilamine, promethazine, chlorpheniramine or chlorcyclizine;    -   a sedative such as glutethimide, meprobamate, methaqualone or        dichloralphenazone;    -   a skeletal muscle relaxant, e.g. baclofen, carisoprodol,        chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine;    -   an NMDA receptor antagonist, e.g. dextromethorphan        ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan        ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,        pyrroloquinoline quinine,        cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine,        EN-3231 (MorphiDex®, a combination formulation of morphine and        dextromethorphan), topiramate, neramexane or perzinfotel        including an NR2B antagonist, e.g. ifenprodil, traxoprodil or        (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone;    -   an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine,        guanfacine, dexmetatomidine, modafinil, terazosin, indoramin,        alfuzosin, silodosin or        4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline;        prazosin    -   a tricyclic antidepressant, e.g. desipramine, imipramine,        amitriptyline or nortriptyline;    -   an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate        or valproate;    -   a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1        antagonist, e.g.        (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione        (TAK-637),        5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one        (MK-869), aprepitant, lanepitant, dapitant or        3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine        (2S,3S);    -   a muscarinic antagonist, e.g oxybutynin, tolterodine,        fesoterodine, 5-hydroxymethyltolterodine, propiverine, trospium        chloride, darifenacin, solifenacin, temiverine and ipratropium;    -   a COX-2 selective inhibitor, e.g. celecoxib, rofecoxib,        parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib;    -   a coal-tar analgesic, in particular acetaminophen/paracetamol;    -   a neuroleptic such as droperidol, chlorpromazine, haloperidol,        perphenazine, thioridazine, mesoridazine, trifluoperazine,        fluphenazine, clozapine, olanzapine, risperidone, ziprasidone,        quetiapine, sertindole, aripiprazole, sonepiprazole,        blonanserin, iloperidone, perospirone, raclopride, zotepine,        bifeprunox, asenapine, lurasidone, amisulpride, balaperidone,        palindore, eplivanserin, osanetant, rimonabant, meclinertant,        Miraxion® or sarizotan;    -   a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist        (e.g. capsazepine);    -   a beta-adrenergic such as propranolol;    -   a local anaesthetic such as mexiletine;    -   a corticosteroid such as dexamethasone;    -   a 5-HT receptor agonist or antagonist (eg pizotifen), and        particularly a 5-HT_(1B/1D) agonist such as eletriptan,        sumatriptan, naratriptan, zolmitriptan or rizatriptan;    -   a 5-HT_(2A) receptor antagonist such as        R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol        (MDL-100907);    -   a cholinergic (nicotinic) analgesic, such as ispronicline        (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine        (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine        (ABT-594) or nicotine;    -   Tramadol (trade mark);    -   a PDE-5 inhibitor, such as        5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (sildenafil),        (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′1′,1:6,1]-pyrido[3,4-b]indole-1,4-dione        (IC-351 or tadalafil),        2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one        (vardenafil),        5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide,        3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide;    -   an alpha-2-delta ligand such as gabapentin, pregabalin,        3-methylgabapentin,        (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid,        (3S,5R)-3-aminomethyl-5-methyl-heptanoic acid,        (3S,5R)-3-amino-5-methyl-heptanoic acid,        (3S,5R)-3-amino-5-methyl-octanoic acid,        (2S,4S)-4-(3-chlorophenoxy)proline,        (2S,4S)-4-(3-fluorobenzyl)-proline,        [(1R,5R,6S)-6-(aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid,        3-(1-aminomethyl-cyclohexylmethyl)-4H-[1,2,4]oxadiazol-5-one,        C-[1-(1H-tetrazol-5-ylmethyl)-cycloheptyl]-methylamine,        (3S,4S)-(1-aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid,        (3S,5R)-3-amino-5-methyl-nonanoic acid,        (3R,4R,5R)-3-amino-4,5-dimethyl-heptanoic acid and        (3R,4R,5R)-3-amino-4,5-dimethyl-octanoic acid;        (3S,5R)-3-aminomethyl-5-methyloctanoic acid;    -   a cannabinoid;    -   metabotropic glutamate subtype 1 receptor (mGluR1) antagonist;    -   a serotonin reuptake inhibitor such as sertraline, sertraline        metabolite desmethylsertraline, fluoxetine, norfluoxetine        (fluoxetine desmethyl metabolite), fluvoxamine, paroxetine,        citalopram, citalopram metabolite desmethylcitalopram,        escitalopram, d,l-fenfluramine, femoxetine, ifoxetine,        cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine        and trazodone;    -   a noradrenaline (norepinephrine) reuptake inhibitor, such as        maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine,        tomoxetine, mianserin, buproprion, buproprion metabolite        hydroxybuproprion, nomifensine and viloxazine (Vivalan®),        especially a selective noradrenaline reuptake inhibitor such as        reboxetine, in particular (S,S)-reboxetine;    -   a dual serotonin-noradrenaline reuptake inhibitor, such as        venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine,        clomipramine, clomipramine metabolite desmethylclomipramine,        duloxetine, milnacipran and imipramine;    -   an inducible nitric oxide synthase (iNOS) inhibitor such as        S-[2-[(1-iminoethyl)amino]ethy]-L-homocysteine,        S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine,        S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,        (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic        acid,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile;        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile,        (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile,        N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine,        or guanidinoethyldisulfide;    -   an acetylcholinesterase inhibitor such as donepezil;    -   a prostaglandin E₂ subtype 4 (EP4) antagonist such as        N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide        or        4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic        acid;    -   a leukotriene B4 antagonist; such as        1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic        acid (CP-105696),        5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric        acid (ONO-4057) or DPC-11870,    -   a 5-lipoxygenase inhibitor, such as zileuton,        6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone        (ZD-2138), or        2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504);    -   a sodium channel blocker, such as lidocaine; or bupivicaine    -   a 5-HT3 antagonist, such as ondansetron;    -   glycosaminoglycan layer replacer and anti-inflammatory, such as        pentosan polysulphate (Elmiron—trade mark);    -   a beta-3 agonist, such as YM-178 (mirabegron or        2-amino-N-[4-[2-[[(2R)-2-hydroxy-2-phenylethyl]amino]ethyl]phenyl]-4-thiazoleacetamide),        solabegron, KUC-7483 (ritobegron or        2-[4-[2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-2,5-dimethylphenoxy]-acetic        acid) or AK-134;    -   an anti-histamine, such as hydroxyzine;    -   a H₂-antagonist, such as cimetidine; or ranitidine    -   silver nitrate;    -   a steroid;    -   doxorubicin;    -   chondroitin sulphate;    -   disodium chromoglycate;    -   oxychlorosene (Clorpactin—trade mark); and    -   an immunosuppressant, such as cyclosporine        and the pharmaceutically acceptable salts and solvates thereof.

Administration of an Anti-NGF Antagonist Antibody

The anti-NGF antagonist antibody can be administered to an individual(for pain and/or lower urinary tract symptoms associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome) via any suitable route. It should be apparent to a personskilled in the art that the examples described herein are not intendedto be limiting but to be illustrative of the techniques available.Accordingly, in some embodiments, the anti-NGF antagonist antibody isadministered to an individual in accord with known methods, such asintravenous administration, e.g., as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, sublingually,intrasynovial, via insufflation, intrathecal, oral, inhalationtransdermal or topical routes. Administration can be systemic, e.g.,intravenous administration, or localized. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, anti-NGF antagonist antibody can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

In one embodiment, an anti-NGF antagonist antibody is administered viasite-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the anti-NGF antagonist antibody or localdelivery catheters, such as infusion catheters, an indwelling catheter,or a needle catheter, synthetic grafts, adventitial wraps, shunts andstents or other implantable devices, site specific carriers, directinjection, or direct application. See, e.g., PCT Publication No. WO00/53211 and U.S. Pat. No. 5,981,568.

Various formulations of antibodies such as E3 or fragments thereof(e.g., Fab, Fab′, F(ab′)2, Fv, Fc, etc.), such as single chain (ScFv),mutants thereof, fusion proteins comprising an antibody portion, and anyother modified configuration that comprises an antigen NGF recognitionsite of the required specificity, may be used for administration. Insome embodiments the anti-NGF antagonist antibody may be administeredneat. In some embodiments, anti-NGF antagonist antibody and apharmaceutically acceptable excipient may be in various formulations.Pharmaceutically acceptable excipients are known in the art, and arerelatively inert substances that facilitate administration of apharmacologically effective substance. For example, an excipient cangive form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

An anti-NGF antibody can be administered using any suitable method,including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Anti-NGF antibodies can also beadministered via inhalation or by other forms of administration (e.g.oral, mucosal, sublingually), as described herein. Generally, foradministration of anti-NGF antibodies, an initial candidate dosage canbe about 2 mg/kg.

In a preferred embodiment, the concentration of the antibody to beadministered can range from about 0.1 to about 200 mg/ml. Preferably theconcentration of antibody is about 0.5 mg/ml, about 1 mg/ml, about 2mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml,about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml,about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25mg/ml, about 26 mg/ml, about 27 mg/ml, about 28 mg/ml, about 29 mg/ml,about 30 mg/ml, about 31 mg/ml, about 32 mg/ml, about 33 mg/ml, about 34mg/ml, about 35 mg/ml, about 36 mg/ml, about 37 mg/ml, about 38 mg/ml,about 39 mg/ml, about 40 mg/ml, about 41 mg/ml, about 42 mg/ml, about 43mg/ml, about 44 mg/ml, about 45 mg/ml, about 46 mg/ml, about 47 mg/ml,about 48 mg/ml, about 49 mg/ml, about 50 mg/ml, about 51 mg/ml, about 52mg/ml, about 53 mg/ml, about 54 mg/ml, about 55 mg/ml, about 56 mg/ml,about 57 mg/ml, about 58 mg/ml, about 59 mg/ml, about 60 mg/ml, about 70mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml or about 110mg/ml.

Most preferably the concentration of antibody is selected from the groupcomprising about 2 mg/ml, about 2.5 mg/ml, about 5 mg/ml, about 10mg/ml, about 20 mg/ml, about 22 mg/ml and about 50 mg/ml.

In some embodiments, the administration pattern of the anti-NGF antibodycomprises administration of a dose once every week, once every 2 weeks,every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 15 weeks,every 20 weeks, every 25 weeks, or longer. In some embodiments, theantibody is administered once every 1 month, every 2 months, every 3months, every 4 months, every 5 months, every 6 months, or longer. Mostpreferably, the anti-NGF antibody is administered once every 8 weeks.The progress of this therapy may be monitored by conventional techniquesand assays. The dosing regimen (including the NGF antagonist(s) used)can vary over time.

In some embodiments the volume of a dose is less than or equal to about20 ml, about 15 ml, about 10 ml, about 5 ml, about 2.5 ml, about 1.5 ml,about 1.0 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or about 0.01ml. In some embodiments the volume of a dose is about 20 ml, about 19ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml,about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2ml or about 1 ml. Alternatively about 20.5 ml, about 19.5 ml, about 18.5ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml,about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml,about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5. Alternativelyabout 900 μl, about 800 μl, about 700 μl, about 600 μl, about 500 μl,about 400 μl, about 300 μl, about 200 μl, or about 100 μl, alternativelyabout 950 μl, about 850 μl, about 750 μl, about 650 μl, about 550 μl,about 450 μl, about 350 μl, about 250 μl, about 150 μl, or about 50 μl.Most preferably the volume of the dose is less than or equal to about2.5 ml.

According to a preferred embodiment the dose contains less than or equalto about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg,about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg,about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg,about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg,about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg,about 70 mg, about 80 mg, about 90 mg, about 100 mg, or about 110 mg ofantibody. Most preferably the dose contains less than or equal to about50 mg of antibody.

According to a preferred embodiment the dose contains an amount ofantibody that is about 1 μg/kg, about 10 μg/kg, about 20 μg/kg, about 50μg/kg, about 100 μg/kg, about 200 μg/kg, about 500 μg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, orabout 11 mg/kg (of mass of the mammal to which the dose it to beadministered).

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toreduce pain. In an embodiment a dosing regimen may compriseadministering an initial dose of about 2 mg/kg, followed by a weeklymaintenance dose of about 1 mg/kg of the anti-NGF antibody, or followedby a maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, in some embodiments, dosing from one-four times a week iscontemplated. Even less frequent dosing may be used.

For the purpose of the present invention, the appropriate dosage of ananti-NGF antagonist antibody will depend on the antibody (orcompositions thereof) employed, the type and severity of the pain orlower urinary tract symptom to be treated, whether the agent isadministered for preventative or therapeutic purposes, previous therapy,the patient's clinical history and response to the agent, and thediscretion of the attending physician. Typically the clinician willadminister an anti-NGF antagonist antibody, until a dosage is reachedthat achieves the desired result. Dose and/or frequency can vary overcourse of treatment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of pain. Alternatively, sustained continuous releaseformulations of anti-NGF antagonist antibodies may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

In some individuals, more than one dose may be required. Frequency ofadministration may be determined and adjusted over the course oftherapy. For example, frequency of administration may be determined oradjusted based on the type and severity of the pain and/or lower urinarytract symptom to be treated, whether the agent is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the agent, and the discretion of theattending physician. Typically the clinician will administer an anti-NGFantagonist antibody (such as E3), until a dosage is reached thatachieves the desired result. In some cases, sustained continuous releaseformulations of E3 antibodies may be appropriate. Various formulationsand devices for achieving sustained release are known in the art.

In one embodiment, dosages for an anti-NGF antagonist antibody may bedetermined empirically in individuals who have been given one or moreadministration(s) of an NGF antagonist. Individuals are givenincremental dosages of an anti-NGF antagonist antibody. To assessefficacy of an anti-NGF antagonist antibody, an indicator of pain orlower urinary tract symptoms may be followed, such as a change in a painnumerical rating scale (NRS), a change in the O'Leary-Sant InterstitialCystitis Symptom Index (ICSI), a change in the O'Leary-Sant InterstitialCystitis Problem Index (ICPI), a change in the Pelvic Pain andUrgency/Frequency (PUF) symptom score and/or changes in micturitionvariables including micturition frequency, nocturnal frequency,incontinence episode frequency, mean volume voided per micturition, meaninterstitial cystitis pain severity, urinary urgency episodes, averagesleep disturbance score, average pain associated with sexual activitypain score, global response assessment, patent reporting treatmentimpact assessment, treatment failure, biomarkers, safety endpointsand/or pharmacokinetic measures. Biomarkers may include NGF,glycoprotein-51 (GP-51), antiproliferative factor (APF) and HB-Epidermalgrowth factor (HB-EGF) amongst others.

Administration of an anti-NGF antagonist antibody in accordance with themethod in the present invention can be continuous or intermittent,depending, for example, upon the recipient's physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an anti-NGF antagonist antibody may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced dose, e.g., either before, during, or after developing painand/or lower urinary tract symptoms associated with interstitialcystitis and/or painful bladder syndrome and/or bladder pain syndrome;before; during; before and after; during and after; before and during;or before, during, and after developing pain and/or lower urinary tractsymptoms associated with interstitial cystitis and/or painful bladdersyndrome and/or bladder pain syndrome.

Therapeutic formulations of the anti-NGF antagonist antibody used inaccordance with the present invention are prepared for storage by mixingan antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000)), in the form of lyophilized formulations or aqueoussolutions. In some embodiments, more than one anti-NGF antagonistantibody may be present. At least one, at least two, at least three, atleast four, at least five different, or more anti-NGF antagonistantibodies can be present. Generally, those anti-NGF antagonistantibodies have complementary activities that do not adversely affecteach other. NGF antagonists can also be used in conjunction with otheragents that serve to enhance and/or complement the effectiveness of theagents.

Pharmaceutically acceptable carriers include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Typically, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,antigen-binding portion thereof, immunoconjuage, or bispecific molecule,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and may comprisebuffers such as phosphate, citrate, acetate and other organic acidsincluding amino acid buffers; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosacchandes,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polysorbate, Tween™, PLURONICS™ or polyethyleneglycol (PEG).

In certain embodiments, the antibodies of the present disclosure may bepresent in a neutral form (including zwitter ionic forms) or as apositively or negatively-charged species. In some cases, the antibodiesmay be complexed with a counterion to form a pharmaceutically acceptablesalt. Thus, the pharmaceutical compounds of the disclosure may includeone or more pharmaceutically acceptable salts.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound (e.g. antibody) anddoes not impart undesired toxicological effects (see e.g., Berge, S. M.,et al. (1977) J. Pharm. Sci. 66:1-19). For example, the term“pharmaceutically acceptable salt” includes a complex comprising one ormore antibodies and one or more counterions, where the counterions arederived from pharmaceutically acceptable inorganic and organic acids andbases.

Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

Furthermore, pharmaceutically acceptable inorganic bases includemetallic ions. Metallic ions include, but are not limited to,appropriate alkali metal salts, alkaline earth metal salts and otherphysiological acceptable metal ions. Salts derived from inorganic basesinclude aluminum, ammonium, calcium, cobalt, nickel, molybdenum,vanadium, manganese, chromium, selenium, tin, copper, ferric, ferrous,lithium, magnesium, manganic salts, manganous, potassium, rubidium,sodium, and zinc, and in their usual valences.

Pharmaceutically acceptable acid addition salts of the antibodies of thepresent disclosure can be prepared from the following acids, including,without limitation formic, acetic, acetamidobenzoic, adipic, ascorbic,boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic,malonic, edetic, ethylsulfuric, fendizoic, metaphosphoric, succinic,glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric,ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic,aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic,lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic,mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic,aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic,mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic,ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic,pantothenic, naphthalenesulfonic, toluenesulfonic,2-hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuricacid monomethyl ester, cyclohexylaminosulfonic, β-hydroxybutyric,glycine, glycylglycine, glutamic, cacodylate, diaminohexanoic,camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal5-phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic,galactaric and galacturonic acids.

Pharmaceutically acceptable organic bases include trimethylamine,diethylamine, N,N′-dibenzylethylenediamine, chloroprocaine, choline,dibenzylamine, diethanolamine, ethylenediamine, meglumine(N-methylglucamine), procaine, cyclic amines, quaternary ammoniumcations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine,butylamine, ethanolamine, ethylenediamine, N-ethylmorpholine,N-ethylpiperidine, ethylglucamine, glucamine, glucosamine, histidine,hydrabamine, imidazole, isopropylamine, methylglucamine, morpholine,piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamineresins, procaine, purines, theobromine, triethylamine, tripropylamine,triethanolamine, tromethamine, methylamine, taurine, cholate,6-amino-2-methyl-2-heptanol, 2-amino-2-methyl-1,3-propanediol,2-amino-2-methyl-1-propanol, aliphatic mono- and dicarboxylic acids,phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromaticacids, aliphatic and aromatic sulfonic acids, strontium, tricine,hydrazine, phenylcyclohexylamine, 2-(N-morpholino)ethanesulfonic acid,bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane,N-(2-acetamido)-2-aminoethanesulfonic acid,1,4-piperazinediethanesulfonic acid,3-morpholino-2-hydroxypropanesulfonic acid,1,3-bis[tris(hydroxymethyl)methylamino]propane,4-morpholinepropanesulfonic acid,4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,4-(N-morpholino)butanesulfonic acid,3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid,2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid,4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid),piperazine-1,4-bis(2-hydroxypropanesulfonic acid)dihydrate,4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid,N,N-bis(2-hydroxyethyl)glycine,N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid),N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid,N-tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid,N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid,2-(cyclohexylamino)ethanesulfonic acid,3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid,3-(cyclohexylamino)-1-propanesulfonic acid, N-(2-acetamido)iminodiaceticacid, 4-(cyclohexylamino)-1-butanesulfonic acid,N-[tris(hydroxymethyl)methyl]glycine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and trometamol.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

Liposomes containing the anti-NGF antagonist antibody are prepared bymethods known in the art, such as described in Epstein, et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad.Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antagonist (such as the antibody),which matrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or ‘poly(v nylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes.

Therapeutic anti-NGF antagonist antibody compositions are generallyplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Therapeutic compositions can be administered with medical devices knownin the art. For example, a therapeutic composition of the disclosure canbe administered with a needleless hypodermic injection device, such asthe devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-knownimplants and modules useful in the present disclosure include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 .lm, particularly 0.1 and 0.5 .lm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an anti NGFantibody) with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Treatment efficacy can be assessed by methods well-known in the art.

A polynucleotide encoding any of the antibodies or polypeptides of theinvention (such as antibody E3) may also be used for delivery andexpression of any of the antibodies or polypeptides of the invention(such as antibody E3) in a desired cell. It is apparent that anexpression vector can be used to direct expression of an E3 antibody orpolypeptide. The expression vector can be administered by any meansknown in the art, such as intraperitoneally, intravenously,intramuscularly, subcutaneously, intrathecally, intraventricularly,orally, enterally, parenterally, intranasally, dermally, sublingually,or by inhalation. For example, administration of expression vectorsincludes local or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions comprising apolynucleotide encoding any of the antibodies or polypeptides of theinvention (such as antibody E3) can also be used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol.Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides of the present inventioncan be delivered using gene delivery vehicles. The gene delivery vehiclecan be of viral or non-viral origin (see generally, Jolly, Cancer GeneTherapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly,Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994)6:148). Expression of such coding sequences can be induced usingendogenous mammalian or heterologous promoters. Expression of the codingsequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP PatentNo. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent NO. 0 524 968.Additional approaches are described in Philip, Mol. Cell Biol. (1994)14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

Diagnostic Methods and Uses

The antibodies described herein may also be used in the detection,diagnosis and monitoring of pain and/or a lower urinary tract symptomassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome associated with altered or aberrant NGFexpression (in some embodiments, increased or decreased NGF expression(relative to a normal sample), and/or inappropriate expression, such aspresence of expression in tissue(s) and/or cell(s) that normally lackNGF expression, or absence of NGF expression in tissue(s) or cell(s)that normally possess NGF expression). In some embodiments, NGFexpression is detected in a sample from an individual suspected ofhaving pain and/or lower urinary tract symptoms associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome.

Thus, in some embodiments, the invention provides methods comprisingcontacting a specimen (sample) of an individual suspected of havingaltered or aberrant NGF expression with an antibody or polypeptide ofthe invention and determining whether the level of NGF differs from thatof a control or comparison specimen.

In other embodiments, the invention provides methods comprisescontacting a specimen (sample) of an individual and determining level ofNGF expression. In some embodiments, the individual is suspected ofhaving pain and/or a lower urinary tract symptom associated withinterstitial cystitis and/or painful bladder syndrome and/or bladderpain syndrome.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety including but not limited to radioisotopes,fluorescent labels, and various enzyme-substrate labels. Methods ofconjugating labels to an antibody are known in the art. In otherembodiments of the invention, antibodies of the invention need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibodies of the invention.

The antibodies of the present invention may be employed in any knownassay method, such competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies may also be used for in vivo diagnostic assays, such asin vivo imaging. Generally, the antibody is labeled with a radionuclide(such as 111In, 99Tc, 14C, 131I, 125I, or 3H) so that the cells ortissue of interest can be localized using immunoscintiography.

The antibody may also be used as staining reagent in pathology,following techniques well known in the art.

With respect to all methods described herein, reference to anti-NGFantagonist antibodies also includes compositions comprising one or moreof these agents. These compositions may further comprise suitableexcipients, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art. The present invention can beused alone or in combination with other conventional methods oftreatment.

Kits Comprising Anti-NGF Antagonist Antibodies for Use in Detectionand/or Therapy

The invention also provides kits comprising antibodies for use indetection and/or therapy. Accordingly, in some embodiments, the kitscomprise an antibody E3. In some embodiments, the kit comprises anyantibody or polypeptide described herein. In other aspects, the kits maybe used for any of the methods described herein, including, for example,to treat an individual with pain and/or a lower urinary tract symptomassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome. The kits of this invention are in suitablepackaging, and may optionally provide additional components such as,buffers and instructions for use of the antibody in any of the methodsdescribed herein. In some embodiments, the kits include instructions fortreating pain or lower urinary tract symptoms. In some embodiments, thekit comprises an anti-NGF antagonist antibody described herein andinstructions for treating and/or preventing pain and/or lower urinarytract symptoms associated with interstitial cystitis and/or painfulbladder syndrome and/or bladder pain syndrome in an individual. In someof the embodiments, the anti-NGF antagonist antibody is antibody E3.

In another aspect, the invention provides kits comprising apolynucleotide encoding an E3 polypeptide as described herein. In someembodiments, the kits further comprise instructions for use of thepolynucleotide in any of the methods described herein.

The following examples are provided to illustrate, but not to limit, theinvention. The Examples in WO2004/058184 are referred to illustrate theantibodies for use in the present invention. The entire content ofWO2004/058184 is hereby incorporated by reference.

EXAMPLES Example 1 Humanization and Affinity Maturation of MouseAntagonist Anti-NGF Antibody 911

A. General Methods

The following general methods were used in this example.

Library Generation

Libraries were generated by PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofpeptides and proteins: a laboratory manual, San Diego, Academic Press(see, pages pg 277-291). The doping codon NNK was used to randomize oneamino acid position to include 20 possible amino acids. To randomize oneamino acid position to include only a subset of amino acids withspecific properties, doping codons were used as described in Balint etal, (1993) Gene 137(1):109-18). Site directed mutagenesis was performedusing recombinant PCR as described in Innis et al, (1990) PCR protocols:A guide to methods and applications (see, pp. 177-183).

Small Scale Fab Preparation

Small scale expression in 96 well plates was optimized for screening Fablibraries. Starting from E. coli transformed with a Fab library,colonies were picked to inoculate both a master plate (agarLB+Ampicillin (50 μg/ml)+2% Glucose) and a working plate (2 ml/well, 96well/plate containing 1.5 mL of LB+Ampicillin (50 μg/ml)+2% Glucose).Both plates were grown at 30° C. for 8-12 hours. The master plate wasstored at 4° C. and the cells from the working plate were pelleted at5000 rpm and resuspended with 1 mL of LB+Ampicillin (50 μg/ml)+1 mM IPTGto induce expression of Fabs. Cells were harvested by centrifugationafter 5 h expression time at 30° C., then resuspended in 500 μL ofbuffer HBS-EP (100 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005% P20, 3 mMEDTA). Lysis of HBS-EP resuspended cells was attained by one cycle offreezing (−80° C.) then thawing at 37° C. Cell lysates were centrifugedat 5000 rpm for 30 min to separate cell debris from supernatantscontaining Fabs. The supernatants were then injected into the BIAcoreplasmon resonance apparatus to obtain affinity information for each Fab.Clones expressing Fabs were rescued from the master plate to sequencethe DNA and for large scale Fab production and detailed characterizationas described below.

Large Scale Fab Preparation

To obtain detailed kinetic parameters, Fabs were expressed and purifiedfrom large cultures. Erlenmeyer flasks containing 200 mL ofLB+Ampicillin (50 μg/ml)+2% Glucose were inoculated with 5 mL of overnight culture from a selected Fab-expressing E. coli clone. Clones wereincubated at 30° C. until an OD_(550 nm) of 1.0 was attained and theninduced by replacing the media for 200 ml, of LB+Ampicillin (50 μg/ml)+1mM IPTG. After 5 h expression time at 30° C., cells were pelleted bycentrifugation, then resuspended in 10 mL PBS (pH 8). Lysis of the cellswas obtained by two cycles of freeze/thaw (at −80° C. and 37° C.,respectively). Supernatant of the cell lysates were loaded onto Ni-NTAsuperflow sepharose (Qiagen, Valencia. CA) columns equilibrated withPBS, pH 8, then washed with 5 column volumes of PBS, pH 8. IndividualFabs eluted in different fractions with PBS (pH 8)+300 mM Imidazol.Fractions containing Fabs were pooled and dialized in PBS, thenquantified by ELISA prior to affinity characterization.

Full Antibody Preparation

For expression of full antibodies, heavy and light chain variableregions were cloned in 2 mammalian expression vectors (Eb.911.E3 orEb.pur.911.3E for light chain and Db.911.3E for heavy chain; describedherein) and transfected using lipofectemine into HEK 293 cells fortransient expression. Antibodies were purified using protein A usingstandard methods.

Biacore Assay

Affinities of anti-NGF Fabs and monoclonal antibodies were determinedusing the BIAcore3000™ surface plasmon resonance (SPR) system (BIAcore,INC, Piscaway N.J.). CM5 chips were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF was diluted into 10 mM sodium acetate pH 4.0 and injected overthe activated chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity were achieved: 100-200 response units (RU) for detailed kineticstudies and 500-600 RU for screening assays. The chip was blocked withethanolamine. Regeneration studies showed that a mixture of Pierceelution buffer (Product No. 21004, Pierce Biotechnology, Rockford, Ill.)and 4 M NaCl (2:1) effectively removed the bound Fab while keeping theactivity of hNGF on the chip for over 200 injections. HBS-EP buffer(0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% Surfactant P29) wasused as running buffer for all the BIAcore assays.

Screening Assay

A screening BIAcore assay was optimized to determine the affinity of Fabclones from libraries. Supernatants of small culture lysates wereinjected at 50 μl/min for 2 min. Dissociation times of 10 to 15 minuteswere used for determination of a single exponential dissociation rate(k_(off)) using BIAevaluation software. Samples that showed k_(off)rates in the same range as the template used to create the library(clone 8L2-6D5, k_(off) 1×10⁻³ s⁻¹) were injected for confirmation anddissociation times of up to 45 min were allowed to obtain better k_(off)values. Clones showing improved (slower) k_(off) values were expressedat large scale and full kinetic parameters, k_(on) and k_(off), weredetermined on purified protein. The assay was capable of detectingdifferences in affinity that were approximately 2-fold or larger.

Affinity Determination Assay

Serial dilutions (0.1-10× estimated K_(D)) of purified Fab samples wereinjected for 1 min at 100 μL/min and dissociation times of up to 2 hwere allowed. The concentrations of the Fab proteins were determined byELISA and/or SDS-PAGE electrophoresis using as a standard a Fab of knownconcentration (as determined by amino acid analysis). Kineticassociation rates (k_(on)) and dissociation rates (k_(off)) wereobtained simultaneously by fitting the data to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values were calculated as k_(off)/k_(on).

B. Humanization and Affinity Maturation of Mouse Antagonist Anti-NGFAntibody 911

The mouse antagonist anti-NGF antibody, 911 (see Hongo et al, (2000)Hybridoma 19(3):215-227) was selected for humanization and affinitymaturation. Mab 911 binds human and rat NGF with high affinity andexhibits no significant cross-reactivity with the neurotrophins NT3,NT4/5 or BDNF. See Hongo, id. The affinity of the papain-cleaved Fabfragment of mouse Mab 911 was determined using BIAcore analysis asdescribed above. The papain-cleaved Fab fragment of mouse Mab 911 boundhuman NGF with a K_(D) of approximately 10 nM.

Humanization and affinity maturation was conducted in several steps, asfollows:

(1) Preparation of CDR-Grafted Template.

The light chain extended CDRs of antibody 911 (i.e., including both theKabat and Chothia CDR regions) were grafted into the human germlineacceptor sequences O8 with JK2 and the heavy chain extended CDRs ofantibody 911 were grafted in to human germline acceptor sequence VH4-59with JH4. The amino acid sequences of the human germline acceptorsequences are shown in FIGS. 1A and 1B of WO2004/058184. Amino acidnumbering is sequential. Using the protein frameworks noted above, DNAsequences were designed for synthetic genes encoding human frameworkwith the murine CDRs. These humanized heavy and light variable domainswere termed hVH and hVL respectively. Codons were optimized for E. coliand hamster usage. Several overlapping oligonucleotides (69-90 bases inlength) extending the full length of the hVL and hVH with two shortflanking primers for each chain were used to separately synthesize thetwo genes by recursive PCR essentially as described in Prodromou et al,(1992) Protein Eng 5(8): 827-9. Resulting DNA fragments of the correctlength were gel purified and then cloned into an E. coli bicistronicexpression plasmid (ampicillin resistant). Expression of the antibodieswas under control of an IPTG inducible lacZ promoter similar to thatdescribed in Barbas (2001) Phage display: a laboratory manual, ColdSpring Harbor, N.Y., Cold Spring Harbor Laboratory Press (see VectorpComb3X, at pg 2.10), however, modifications included addition andexpression of the following additional domains: the human Kappa lightchain constant domain (see GenBank Accession No. CAA09181) and the CHIconstant domain of IgG2a human immunoglobulin (GenBank Accession No.P01859).

The amino acid sequences of the variable regions of the CDR-graftedantibody (also termed the “template”), termed 8L2-4D5, are also shown inFIGS. 1A and 1B of WO2004/058184. The affinity of 8L2-4D5 was determinedusing BIAcore analysis as described above. 8L2-4D5 bound human NGF witha K_(D) of approximately 38 nM.

(2) Introduction of a Point Mutation into the Framework Sequence.

The V71K substitution was introduced into the CDR-grafted heavy chainusing recombinant PCR site directed mutagenesis as described in Innis etal, (1995) PCR strategies, San Diego, Academic Press. This substitutionreplaced the human framework residue with the corresponding mouseframework residue. The resulting antibody was termed 8L2-6D5, and theamino acid sequence of the heavy chain variable region of 8L2-6D5 isshown in FIG. 1A of WO2004/058184. The affinity of 8L2-6D5 wasdetermined using BIAcore analysis as described above. The Fab fragmentof 8L2-6D5 bound human NGF with a Kd of approximately 15 nM. 8L2-6D5 waschosen as template for affinity maturation.

(3) Humanization and Affinity Maturation of CDRs L1, L2, H1 and H2.

CDRs L1, L2, H1 and H2 were subjected to humanization and affinitymaturation. Amino acid positions in CDRs L1, L2, H1, and H2 wereidentified that are not essential for the structure of the CDRs based onthe Chothia canonical structure (see Al-Lazikani et al (1997) J. Mol.Biol. 273(4):927-48); and subjected to randomization as follows. Twolibraries were prepared containing the light chain mutations or heavychain mutations shown in Table 2, and the grafted (mouse) CDR L3 or CDRH3, respectively, using PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofpeptides and proteins: a laboratory manual, San Diego, Academic Press,using doping codons as described in Balint et al, (1993) Gene137(1):109-18). Generally, the amino acid residues were altered toresidues that are more common in human antibodies, based on alignmentsof antibody 911 light chain and heavy chain amino acid sequences withhuman germline antibody sequences. The wildtype (unsubstituted) aminoacid residue was also represented in the library with the exception ofCDR H2 residue 50, a methionine, in which the wildtype methionine wasnot represented in the library. Methionine residues are subject tooxidation; thus, replacement of that residue was expected to improvestability of the resulting antibody. The libraries of Fabs were clonedinto vector pComb3X plus the human CH1 and CK regions, as describedabove.

TABLE 2 1. Heavy chain H1/H2 library: CDR-H1 I34 was changed to F, L, V,S, P, T, A, or I N35 was changed to N, T, S, or Y CDR-H2 M50 was changedto all 20 natural amino acids A62 was changed to A or S L63 was changedto L or V 2. Light chain L1/L2 library CDR-L1 S26 was changed to S, A,V, or F D28 was changed to D, A, S, or Y H32 was changed to H, N, K, D,E, Q, or Y CDR-L2 Y50 was changed to Y, D, A, or S I51 was changed to I,T, A, or V F54 was changed to F or L S56 was changed to S and T

For affinity screening experiments, each library was further paired withthe corresponding CDR-grafted light or heavy chain (for example, theH1/H2 library was paired with CDR-grafted light chain), the antibody wasexpressed, and affinity to human NGF of the individual clones wasscreened using the BIACORE surface plasmon resonance (SPR) system(BIAcore, Inc. Piscataway, N.J.) according to the manufacturer'sinstructions and as described above. k_(off), k_(on) and K_(D) weredetermined. Antibody clones were ranked based on k_(off) rates, sincegenerally most variation in affinity is seen in k_(off) rates, andfurther because k_(off) rates are independent of antibody concentration.

The sequence of clones that bound was determined and the sequence ofclones that bound is shown in table 3.

TABLE 3 L1 and L2 amino acid sequences, H1 and H2 amino acidsequences, and kinetic data for clones that boundfollowing affinity screening of H1/H2 or L1/L2 library clones. CDR 1-2mutants kinetic data Light chain library clones Paired with 8L2 heavyCDRL1 CDRL2 k_(off) *K_(D) chain AA sequence AA sequence (s−1) (nM)8L2-6D5 RASQDISNHLN YISRFHS **1e−3 25 (control) (SEQ ID NO: 12)(SEQ ID NO: 13) L129 RASQSISNNLN YTSRFHS 4.5e−4 11 (SEQ ID NO: 18)(SEQ ID NO: 19) L208 RASQYISNHLN YTSRFHS 4.6e−4 11 (SEQ ID NO: 20)(SEQ ID NO: 21) L97 RASQSISNQLN YVSRFHS 5.6e−4 14 (SEQ ID NO: 22)(SEQ ID NO: 23) L81 RAFQAISNQLN YISRFHT 7.4e−4 18 (SEQ ID NO: 24)(SEQ ID NO: 25) L6 RAFQSISNQLN YASRFHS 8.2e−4 20 (SEQ ID NO: 26)(SEQ ID NO: 27) Heavy chain library clones Paired with 6D5 Light CDRH1CDRH2 k_(off) *K_(D) chain AA sequence AA sequence (s−1) (nM) 8L2-6D5GFSLIGYDIN MIWGDGTTDYNSAL   1e−3 25 (control) (SEQ ID NO: 9)(SEQ ID NO: 10) H109 GFSLIGYDSN IIWGDGTTDYNSAL 1.6e−4 4 (SEQ ID NO: 28)(SEQ ID NO: 29) H19 GFSLIGYDLN IIWGDGTTDYNSAV 2.4e−4 6 (SEQ ID NO: 30)(SEQ ID NO: 31) H222 GFSLIGYDVT GIWGDGTTDYNSAV 3.8e−4 9.5(SEQ ID NO: 32) (SEQ ID NO: 33) H225 GFSLIGYDVT GIWGDGTTDYNSSV 3.8e−49.5 (SEQ ID NO: 34) (SEQ ID NO: 35) H18 GFSLIGYDAT GIWGDGTTDYNSAV 4.2e−410.5 (SEQ ID NO: 36) (SEQ ID NO: 37) H9 GFSLIGYDVS IIWGDGTTDYNSSV 4.1e−410.2 (SEQ ID NO: 38) (SEQ ID NO: 39) H227 GFSLIGYDIS QIWGDGTTDYNSSV5.4e−4 13.5 (SEQ ID NO: 40) (SEQ ID NO: 41) H17 GFSLIGYDASGIWGDGTTDYNSSV 6.1e−4 15.2 (SEQ ID NO: 42) (SEQ ID NO: 43) H28GFSLIGYDST SIWGDGTTDYNSAL 7.5e−4 18.7 (SEQ ID NO: 44) (SEQ ID NO: 45) AAin bold were randomized as indicated above *KD calculated using k_(on)4e4 M⁻¹s⁻¹ **For convenience, “e” as used herein denotes “x10.” Thus,4e4 interchangeably means 4 × 10⁴.

CDRs containing the following substitutions retained binding:

-   -   CDR-H1        -   I34: S, L, V, I and A bound.        -   N35: N, T and S bound.    -   CDR-H2        -   M50: M, I, G, Q, S, L bound.        -   A62: A and S bound.        -   L63: L and V bound.    -   CDR-L1        -   S26: S, and F bound.        -   D28: D, S, A, Y bound.        -   H32: H, N, Q bound.    -   CDR-L2        -   Y50: Y bound.        -   I51: I, T, V, A, bound.        -   F54: F bound        -   S56: S and T bound

CDRs containing the following substitutions were selected generallybased on binding affinity and combined into a single clone, termedH19-L129:

-   -   CDR-H1: I34L; N35N (no change)    -   CDR-H2: M50I; A62A (no change); L63V    -   CDR-L1: S26S (no change); D28S; H32N    -   CDR-L2: Y50Y (no change); I51 T; F54F (no change); S56S (no        change)

These mutations were combined (by amplifying the H and L chains by PCR,cutting the PCR products and vector (pRN8) with restriction enzyme andperforming a 3 fragment ligation) into a single clone, termed H19-L129,which also included the grafted H3 and L3 CDRs. The sequence of theheavy chain and light chain variable regions of H19-L129 is shown inFIGS. 1A and 1B of WO2004/058184, and Table 4 shows the amino acidsequence of CDRs L1, L2, H1 and H2. H19-L129 bound NGF with a KD ofapproximately 1 nM, as determined using BIAcore analysis as describedherein.

TABLE 4 Amino acid sequence of CDRs H1, H2, L1 and L2 and kineticdata for combined clone H19-L129. Combination clone: mutations in CDRL1CDRL2 CDRs H1, H2, CDRH1 CDRH2 k_(off) *K_(D) L1, L2 AA sequenceAA sequence (s−1) (nM) H19-L129 CDR-L1: CDRL2: 1.1e−4 3.5 RASQSISNNLNYTSRFHS (SEQ ID NO: 18) (SEQ ID NO: 19) CDR H1: CDR-H2: GFSLIGYDLNIIWGDGTTDYNSAV (SEQ ID NO: 30) (SEQ ID NO: 31) *KD calculated usingk_(on) 4e4 M⁻¹s⁻¹

(4) Affinity Maturation of H3 and L3 CDRs.

Affinity maturation of the H3 and L3 CDRs was carried out in two steps.First, in a process termed “library scanning mutagenesis”, each aminoacid residue in H3 and L3 was individually prescreened in order toidentify amino acid positions at which a mutation resulted in increasedbinding affinity to human NGF. Based on the results of the libraryscanning mutagenesis (also termed “small library randomizationanalysis”), a subset of amino acid positions in H3 and L3 were selectedfor preparation of the affinity maturation library, and the affinitymaturation library was screened for affinity to human NGF using BIAcoreanalysis as described herein. It is appreciated that these techniquescan be generally applied.

(a) Library Scanning Mutagenesis

Each amino acid position in the H3 and L3 CDRs was individuallypre-screened for substitutions which resulted in increased bindingaffinity to human NGF. The frequency of amino acid substitutions at anygiven position that resulted in improved binding, the same binding,worse binding or no binding provided information relating to relating topositions in the CDRs that can be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which cannotbe changed or which can only be changed to a few amino acids. Amino acidsubstitutions resulting in increased binding affinity were alsoidentified. Based on the results of this screening, a subset of aminoacid positions in CDRs H3 and L3 were selected for preparation of anaffinity maturation library.

Individual Fab libraries were prepared in which each amino acid of L3and H3 CDRs was randomized to all 20 amino acids, one at a time,resulting in several (5 libraries for the light chain and 13 librariesfor the heavy chain) small libraries, each with a complexity of 20 aminoacid possibilities at each amino acid position. In all cases, the native(i.e., unchanged) amino acid was represented in the library. Librarieswere prepared by PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofPeptides and Proteins: a laboratory manual, San Diego, Academic Press,using the doping codon NNK to randomize one amino acid position toinclude 20 possible amino acids. The 8L2-6D5 (the CDR grafted antibody,having the framework mutation V71K) served as the template for libraryconstruction because the lower affinity of the CDR grafted antibodypermitted easier detection of differences in affinity in H3 and L3mutants during screening. Thus, each member of a library contained aCDR3 (either H3 or L3) with one amino acid substitution, and 5 graftedCDRs.

20-80 clones from each small library were screened using BIAcoreanalysis as described herein. Samples were simultaneously analyzed byBIAcore for binding affinity to NGF in one channel of the BIAcore chipand for presence of Fab by binding to a penta-histag antibody in anotherchannel of the sensor chip, to detect the his tag at the C terminus ofthe heavy chain. Clones that expressed protein were classified as havingthe same affinity, worse affinity, better affinity or no binding, usingkoff to classify: The results of this analysis are shown in Table 5.

TABLE 5 Clones that expressed protein were classified as having the sameaffinity, worse affinity, better affinity or no binding, based on koff.Percentage of AAs that same retain better ≧1e−3, Worse binding mutation1e−3< 2e−3< ≧2e−3 no bind capacity Light L_S91X 13% 40% 20% 26% 50%chain L_K92X 100% ~100% L_T93X 93%  7% 93% L_L94X 40% 60% 40% L_Y96X 13%80% 7% 13% Heavy H_G98X 50% 37% 13% 50% chain H_G99X 46% 54% 46% H Y100X26% 73% 26% H Y101X 6% 12% 82% 6% H_Y102X 7% 25 68% 7% H_G103X 4% 21%16% 58% 25% H_T104X 20% 30% 50% 20% H_S105X 10% 25% 26% 39% 35% H_Y106X75% 25% 75% H_Y107X 8% 46% 46% 8% H_F108X 23% 27% 50% 23% H_D109X 29%46% 25% 29% H_Y110X 90%  5% 5% 90%

The sequence of all clones with improved affinity was determined,revealing the frequency and identity of amino acid substitutions thatresulted in increased affinity. In addition, a few clones that retainedan affinity similar to the 8I2-6D5 clone were selected from eachlibrary, in order to ascertain amino acid sequence substitutions thatwere permitted at a given position, even though the substitution did notnecessarily increase binding affinity. The results of this analysis aresummarized in Table 6.

TABLE 6 CDR H3 mutations (8L2-6D5 template, including antibody911 CDR-H3 amino acid sequence: GGYYYGTSYYFDY k_(off) (s−1) K_(D)* (nM)(SEQ ID NO: 11) 1E−3 25 Y100L  1.2E−3 30 Y100R  1.1E−3 27 Y101W  5.6E−414 G103A  1.6E−4 4 T104S  2.2E−3 55 S105A  5.1E−4 13 S105T  6.4E−4 16Y106R  1.6E−3 40 Y106T  2.0E−3 50 Y106M  2.7E−3 67 Y107F  1.4E−3 35F108W 1.22E−3 30 D109N  1.5E−3 37 D109G    1E−3 25 Y110K  1.4E−3 35Y110S  1.5E−3 37 Y110R  1.6E−3 40 Y110T  1.7E−3 42 CDR L3 mutations(8L2-6D5 template, including wildtype (unsubstituted) CDR-L3 amino acidsequence: QQSKTLPYT k_(off) (s−1) K_(D)* (nM) (SEQ ID NO:⁻14) 1E−3 25S91E  2.5E−4 6 Y96R  1.7E−3 42 *KD calculated using k_(on) 4e4 M⁻¹s⁻¹

Several mutations resulted in increased binding affinity. At least thefollowing mutations resulted in significantly increased binding affinityas compared with the 8L2-6D5 template: (H_Y101W (CDR sequenceGGYWYGTSYYFDY (SEQ ID NO:46)); H_S105A (CDR sequence GGYYYGTAYYFDY (SEQID NO:47)); H_S105T (CDR sequence GGYYYGTTYYFDY (SEQ ID NO:48)); H_G103A(CDR sequence GGYYYATSYYFDY (SEQ ID NO:49); and L_S91 E (CDR sequenceQQEKTLPYT (SEQ ID NO:50)).

The results of this experiment were used to guide selection of aminoacid positions for generation of the affinity maturation libraries.

This experiment also provided information regarding the frequency ofamino acid substitutions at any given position that resulted in improvedbinding, the same binding, worse binding or no binding, as shown inTable 5. This information permitted identification of amino acidpositions in the CDRs that could be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which could bechanged to a few amino acids or a very few amino acids (in someembodiments, no amino acids). These results also demonstrated amino acidsubstitutions that increased binding affinity.

(b) Affinity Maturation

Next, the results of the small library randomization analysis (above)were used to select residues for production of the H3 and L3 librariesfor affinity maturation of the H3 and L3 CDRs. Residues Y101 and G103 ofCDR H3 and residues S91 and K92 of CDR L3 were selected for productionof the H3 and L3 libraries for affinity maturation of the H3 and L3CDRs.

This library combined mutations in H3 and L3 at the same time inCDR-grafted clone 8L2-6D5, and separately in the background of H19-L129,and had a diversity of 80 different clones. Table 7 shows the amino acidresidues selected for substitution and the amino acids that weresubstituted at each position.

TABLE 7 Amino acid residues in H3 and L3 selected for substitution andthe amino acids that were substituted at each position CDR-H3: Y101 waschanged to Y and W, C. (Note that C was included because use of codonTRS in one degenerated oligonucleotide also generated codon C). G103 waschanged to A, P, S CDR-L3: S91 was changed to E. K92 was changed to alltwenty amino acids. A, R, K, and H bound.

Each polypeptide was expressed as a Fab, and affinity to human NGF of 96individual clones was screened for each library using BIACORE analysisaccording to the manufacturer's instructions and described above. Theresults of this analysis are shown in Table 8.

TABLE 8 CDR L3 H3 COMBINATION mutations k_(off) (s−1) K_(D)* (nM)(8L2-6D5 template) 1E−3 25 L_S91E; L_K92A 5.5E−4 13(CDR sequence QQEATLPYT (SEQ ID NO: 51)) H_Y101W; H_G103A(CDR sequence GGYWYATSYYFDY (SEQ ID NO: 52)) L_S91E; L_K92R 1.0E−4 25(CDR sequence QQERTLPYT (SEQ ID NO: 53)) H_Y101W; H_G103A(CDR sequence GGYWYATSYYFDY (SEQ ID NO: 54))CDR L3 H3 COMBINATION mutations (H19-L129 template, H1H2L1L2k_(off) (s−1) matured) 1.1e−4 K_(D)* (nM) L_S91E; L_K92H 1.2E−5 0.3(CDR sequence QQEHTLPYT (SEQ ID NO: 55)) H_Y101W; H_G103A(CDR sequence GGYWYATSYYFDY (SEQ ID NO: 56)) (CLONE E3) L_S91E; L_K92S4.7E−5 1.1 (CDR sequence QQESTLPYT (SEQ ID NO: 57)) H_Y101W; H_G103S(CDR sequence GGYWYSTSYYFDY (SEQ ID NO: 58)) L_S91E; L_K92K   2E−5 0.5(CDR sequence QQEKTLPYT (SEQ ID NO: 59)) H_Y101Y; H_G103A(CDR sequence GGYYYATSYYFDY (SEQ ID NO: 60)) L_S91E; L_K92R 1.4E−5 0.35(CDR sequence QQERTLPYT (SEQ ID NO: 61)) H_Y101W; H_G103A(CDR sequence GGYWYATSYYFDY (SEQ ID NO: 62)) (CLONE 3C) L_S91E; L_K92R1.5E−5 0.37 (CDR sequence QQERTLPYT (SEQ ID NO: 63)) H_Y101Y; H_G103A(CDR sequence GGYYYATSYYFDY (SEQ ID NO: 64)) *KD calculated using k_(on)4e4 M⁻¹s⁻¹

Based on binding affinity, the best clones, E3 (interchangeably termed“3E”) and 3C, were selected for further characterization. E3 comprisedthe following CDR substitutions: CDR-H3: Y101W, G103A; and CDR-L3: S91E,K92H, which were combined into a single clone which also included thefollowing L1, L2, H1 and H2 mutations:

-   -   CDR-H1: I34L;    -   CDR-H2: M50I; L63V;    -   CDR-L1: D28S; H32N;    -   CDR-L2: I51T.

The sequence of the heavy chain and light chain variable regions of E3is shown in SEQ ID Nos. 1 and 2 (see also FIGS. 1A and 1B ofWO2004/058184). 3C comprised the following CDR substitutions: CDR-L3:S91E; K92R; CDRH3: Y101W; G103A, which were combined into a single clonewhich also included the L1, L2, H1 and H2 mutations described for clone3E.

3E and 3C sequences were cloned into mammalian expression vectors forproduction of Fab and full antibody, and expressed in HEK293 cells andpurified using Ni-NTA or protein A chromatography. Pure protein wasaccurately quantified by amino acid analysis.

The binding affinities to human NGF of Fabs E3 and 3C were measuredusing BIAcore analysis according to the manufacturer's instructions andas described above, except that 100 RU NGF was used on chip to prevent arebinding effect. Briefly, several concentrations of antibodies (Fabs)were injected for 2 minutes onto a CM5 chip with 100 RU of immobilizedhuman NGF on it, and permitted to dissociate for 1800 seconds. Mouseantibody 911 (Fab) was analyzed as a control. Data was analyzed usingBIAevaluation software following the manufacturer's instructions. Theresults of the analysis of antibody E3 and 911 are shown in FIGS. 9 and10 of WO2004/058184. E3 bound human NGF with a KD of approximately 0.07nM (and with a kon of about 6.0e5 M-1s-1, and a k_(off) of about 4.2e-5s-1). 3C bound human NGF with a KD of approximately 0.35 nM (with ak_(off) of about 1.4E-5). By contrast, mouse antibody 911 bound NGF witha KD of 3.7 nM, k_(off) of 8.4×10⁻⁵s⁻¹ and k_(on) of 2.2×10⁴ Ms⁻¹.

Antibody E3 (interchangeably termed 3E) was selected for furtheranalysis based on the high binding affinity. To test the ability of E3to prevent the interaction of NGF with the NGF receptors trkA and p75,2.5 nM of human NGF was premixed and incubated for one hour with 0 to 50nM of antibody E3 (Fab). After the incubation, samples were injected at10 ul/minute on a BIAcore CM5 chip containing 260 RU of p75 (channel 2)and 600 RU of trkA (channel 3), and percent binding was determined. Theresults of this analysis are shown in FIG. 11 of WO2004/058184.Increased concentrations of Fab E3 blocked the interaction of NGF withboth p75 and trkA, as shown by decreased signal (measured in RU),indicating that Fab E3 blocks the interaction of human NGF with bothtrkA and p75. When antibody E3 (Fab) concentration equaled NGFconcentration (at about 2.5 nM NGF concentration), no NGF binding wasobserved (as shown by a signal of zero). The fact that zero percentNGF-receptor binding occurred when concentration of NGF was equal toantibody 3E concentration suggested that 2.5 nM NGF was at leastten-fold higher than the kD of E3 for NGF and at equilibrium.

Example 2 Evaluation of NGF-Blocking Ability of Anti-NGF Antibodiesusing Mouse E13.5 Trigeminal Neuron Survival Assay

The ability of Fab E3 or full antibody E3 to block NGF activity wasevaluated by measurement of the capacity of the antibody to inhibitNGF-dependent survival of mouse E13.5 trigeminal neurons in vitro. Thetrigeminal ganglion is comprised of cutaneous sensory neurons thatinnervate the facial region. The survival of mouse E13.5 trigeminalneurons is a sensitive assay to evaluate the NGF-blocking activity ofanti-NGF antagonist antibodies because NGF is required to supportsurvival of these neurons. For example, at saturating concentrations ofNGF, the survival is close to 100% by 48 hours in culture. By contrast,less than 5% of the neurons survive by 48 hours in absence of NGF.

The survival assay was conducted as follows: time-mated pregnant SwissWebster female mice were euthanised by CO2 inhalation. The uterine hornswere removed and the embryos at embryonic stage E13.5 were extracted anddecapitated. The trigeminal ganglia were dissected usingelectrolytically sharpened tungsten needles. The ganglia were thentrypsinized, mechanically dissociated and plated at a density of 200-300cells per well in defined, serum-free medium in 96-well plates coatedwith poly-L-ornithine and laminin.

The blocking activity of anti-NGF Fabs or antibodies was assessed byadding to the trigeminal neurons varying doses of anti-NGF antibodiesMab 911 (Fab), 8L2-6D5; H19-L129; E3 and 3C; and human or rat NGF at thefollowing concentrations: 0.4 ng/ml (˜15 pM; this concentrationrepresented a saturating concentration of NGF for survival) and 0.04ng/ml (˜1.5 pM; this concentration is around the IC50). After 48 hoursin culture, the cells were subjected to an automated immunocytochemistryprotocol performed on a Biomek FX liquid handling workstation (BeckmanCoulter) as follows: fixation using 4% formaldehyde, 5% sucrose, andPBS; permeabilization using 0.3% Triton X-100 in PBS); blocking ofunspecific binding sites using 5% normal goat serum, 0.11% BSA in PBS;and sequential incubation with a primary and secondary antibodies todetect neurons. The primary antibody was rabbit polyclonal antibodyagainst the protein gene product 89.5 (PGP9.5, Chemicon), an establishedneuronal phenotypic marker. The secondary antibody was Alexa Fluor 488goat anti-rabbit (Molecular Probes), together with the nuclear dyeHoechst 33342 (Molecular Probes) to label the nuclei of all the cellspresent in the culture. Image acquisition and image analysis wereperformed on a Discovery-I/GenII Imager (Universal Imaging Corporation).Images were automatically acquired at two wavelengths for Alexa Fluor488 and Hoechst 33342, with the nuclear staining being used as referencepoint for the image-based auto-focus system of the Imager, since nuclearstaining is present in all of the wells. Appropriate objectives andnumber of sites imaged per well were selected to cover the entiresurface of each well. Automated image analysis was set up to count thenumber of neurons present in each well after 48 hours in culture basedon their specific staining with the anti-PGP9.5 antibody. Carefulthresholding of the image and application of morphology and fluorescenceintensity based selectivity filter resulted in an accurate count ofneurons per well.

The results of this experiment demonstrated that Fab E3 blocked NGFactivity with a high affinity. The results are shown in FIGS. 4-6 ofWO2004/058184, and Table 9.

FIG. 4 of WO2004/058184 is a graph showing NGF-dependent survival ofE13.5 neurons in the presence of varying concentration of human and ratNGF.

FIG. 5 of WO2004/058184 is a graph comparing the NGF blocking effect ofvarious Fabs in the presence of either 0.04 ng/ml of human NGF(approximately 1.5 pM; shown in the lower panel) or 0.4 ng/ml human NGF(approximately 15 pM; shown in the upper panel). 1.5 pM of NGF wasaround the EC50 of NGF promoting survival, while 15 pM represented asaturating concentration of NGF. Survival of E13.5 mouse trigeminalneurons in various concentrations of Fab E3; murine 911 Fab; and FabH19-L129 and Fab 8L2-6D5 was assessed as described above. The IC50 (inpM) was calculated for each Fab at each NGF concentration, and is shownin Table 9. Fab E3 strongly blocked human NGF-dependent trigeminalneuron survival, with an IC50 of approximately 21 pM in the presence of15 pM human NGF, and an IC50 of approximately 1.2 pM in the presence of1.5 pM human NGF. Fabs 3C and H19-L129 also strongly blocked humanNGF-dependent trigeminal neuron survival. FIG. 6 is a graph comparingthe NGF blocking effect of various Fabs in the presence of either 0.04ng/ml of rat NGF (approximately 1.5 pM; shown in the lower panel) or 0.4ng/ml rat NGF (approximately 15 pM; shown in the upper panel). 1.5 pM ofNGF was around the EC50, while 15 pM represented a saturatingconcentration of NGF. Survival of E13.5 mouse trigeminal neurons invarious concentrations of Fab E3; murine Fab 911; and Fab H19-L129 and8L2-6D5 was assessed as described above. The EC50 (in pM) was calculatedfor each Fab at each NGF concentration, and is shown in Table 9. Fab E3strongly blocked human NGF-dependent trigeminal neuron survival, with anIC50 of approximately 31.6 pM in the presence of 15 pM rat NGF, and anIC50 of approximately 1.3 pM in the presence of 1.5 pM rat NGF. Fabs 3Cand H19-L129 also strongly blocked rat NGF-dependent trigeminal neuronsurvival.

TABLE 9 IC50 (in the IC50 (in the presence presence of 15 pM NGF) of 1.5pM NGF) Human NGF pM pM 8L2-6D5 Fab 1580.5 461.8 H19-L129 Fab 60.1 9.63E Fab <21.0 <1.2 3C Fab 80.9 5.6 911 Fab 322.3 63.5 IC50 (15 pM NGF)IC50 (1.5 pM NGF) Rat NGF pM pM 8L2-6D5 Fab 730.3 169.4 H19-L129 Fab31.0 6.0 3E Fab <8.3 <1.3 3C Fab 31.6 6.0 911 Fab 161.0 34.6

In a different experiment, we compared the ability of full antibody E3and Fab 3E to inhibit NGF-dependent survival of E13.5 neurons in thepresence of 0.4 ng/ml saturating concentration) of human NGF. Theresults of the analysis are shown in FIG. 10 of WO2004/058184. Fullantibody E3 and Fab 3E showed similar levels of inhibition ofNGF-dependent survival when the concentration of whole antibody and Fabwere normalized to the number of NGF binding sites (Fab has one bindingsite and whole antibody has two binding sites). These resultsdemonstrated that there was no avidity effect due to the binding of afull antibody to the NGF dimer.

In another experiments, we compared the ability of variousconcentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.0 nM) ofantibody E3, antibody 911, and a trkA receptor immunadhesin (consistingof the extracellular domain of the NGF receptor trkA fused with theimmunoglobulin Fc domain, CH2-CH3) to inhibit NGF-dependent survival ofE13.5 neurons in the presence of 0.4 ng/ml (saturating conditions).These results are shown in FIG. 11 of WO2004/058184. These resultsdemonstrated that antibody E3 blocked NGF better than either antibody911 or the trkA immunoadhesin.

Example 3 Evaluation of the Specificity of Anti-NGF Antibody E3 UsingMouse Trigeminal and Nodose Neuron Survival Assays

The ability of antibody E3 to specifically block NGF activity wasevaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse E17/18 trigeminal neurons in vitro in the presence ofsaturating concentrations of NGF, the NGF-related neurotrophin NT3, orthe NGF-unrelated neurotrophic factor, macrophage stimulating protein(MSP). The survival of mouse E17/18 trigeminal neurons is a sensitiveassay to evaluate the NGF-blocking activity of anti-NGF antagonistantibodies because NGF is required to support survival of these neuronsat higher concentrations than the level of NGF required to supportsurvival of E13.5 TG neurons. Survival of these neurons is alsosupported by NT3 or MSP; therefore, the survival of these neurons isalso a sensitive assay to evaluate whether the anti-NGF antagonistantibody also blocked NT3 or MSP.

The ability of antibody E3 to specifically block NGF activity was alsoevaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse nodose E17 neurons in the presence of saturatingconcentrations of BDNF or NT4/5. Survival of nodose neurons is supportedby BDNF or NT4/5; therefore, survival of these neurons is a sensitiveassay to evaluate the BDNF or NT4/5-blocking ability of the anti-NGFantagonist antibody.

The survival assay was conducted as follows: time mated pregnant SwissWebster female mice were euthanised by CO2 inhalation. The uterine hornswere removed and the embryos (at embryonic day 17 or 18) were extractedand decapitated. The trigeminal and nodose ganglia were dissected andcleaned. The ganglia were then trypsinised, mechanically dissociated andplated at a density of 100-300 cells per well in defined, serum-freemedium in 4-well plates (Greiner) coated with poly-L-ornithine andlaminin.

E17/18 trigeminal neurons were grown either without added neurotrophicfactors (negative control) or in the presence of saturatingconcentrations of human NGF (400 pM and 15 pM) (positive control); NT3(400 pM); or MSP (600 pM). Duplicate cultures were set up that includedvarying concentrations of E3 and 911 Fabs and full antibodies.Concentration of Fab and full antibodies was indicated per binding site(e.g., a full antibody contains two binding sites, while a Fab containsone binding site).

E17 nodose neurons were grown either in the absence of addedneurotrophic factors (negative control), or with saturatingconcentrations of BDNF (400 pM) (positive control) or NT4/5 (400 pM) orNGF unrelated growth factor ILF (interleukin inhibitory factor). Highconcentrations of neurotrophins were used, as the goal of thisexperiment was to test specificity of the antibodies. Duplicate cultureswere set up that included varying again with and without the addition ofantibodies E3 and 911. After 48 hours in culture the total number ofneurons surviving in each well under each condition was ascertained bymanual counting using a phase-contrast microscope.

The results of these experiments demonstrated that E3 and 911 antibodiescompletely blocked the survival promoting effects of NGF on E18trigeminal neurons. By contrast, E3 and 911 antibodies had no effect onsurvival of trigeminal neurons promoted by NT3 or MSP, or survival ofnodose neurons promoted by BDNF or NT4/5 or LIF. These resultsdemonstrated that antibody E3 possessed selective specificity for NGF,as there was no detected interaction between these antibodies and otherNGF related neurotrophins (NT3, NT4/5, BDNF) at concentrations 1000-foldto 10,000-fold higher than effective concentration for NGF blocking.Further, these results demonstrated that the neuronal death seen inNGF-supplemented cultures of NGF-dependent neurons on addition ofantibody or Fab E3 was due to a specific interaction between theseantibodies and NGF and was not due to a generalized toxic effect. Mouseanti-NGF antagonist antibody 911 was also tested, and similar resultswere observed. Note that due to the high concentrations of neurotrophinsused, both antibody E3 and 911 are very close to their titrationconditions and were expected to bind NGF at similar levels because thedifferences in binding affinity of these antibodies to NGF would to beless apparent under these conditions.

The results of these experiments are shown in FIGS. 12, 13, 14, and 15of WO2004/058184. The data showed mean percent survival after 48 hoursin culture (±standard error of mean, n=3 for each data point) relativeto the survival seen in the positive control for each experiment (e.g.,100% survival of trigeminal neurons grown in the presence of saturatingNGF concentration, and 100% survival of nodose neurons grown in thepresence of saturating BDNF concentration, respectively). FIGS. 12-13 ofWO2004/058184 are graphs showing that anti-NGF antagonist antibody E3 orFab E3 did not inhibit the survival promoted by NT3, and MSP, even atantibody concentrations as high as 200 nM. By contrast, 20 nM ofantibody E3 or Fab 3E and Fab 911 totally blocked NGF-elicited survival.Mouse anti-NGF antagonist antibody 911 was also tested, and similarresults were observed. Specifically, FIG. 12 of WO2004/058184 is a graphshowing comparison of the effect of various concentrations (20 nM, 2 nM,or 0.2 nM) of E3 Fab (termed “3E” in the figure) and mouse antibody 911Fab on survival of E18 trigeminal neurons in the presence of no addedneurotrophin (termed “control”), 400 pM NGF (termed “NGF-400 pM), 10 nMNT3 (termed “NT3-10 nM) or 600 pM MSP (termed “MSP-600 pM). FIG. 13 ofWO2004/058184 is a graph depicting comparison of the effect of variousconcentrations (200 nM and 80 nM) of E3 Fab and full antibody and mouseantibody 911 full antibody and Fab of survival of E17 trigeminal neuronsin the presence of no added neurotrophins (termed “no factor”), 400 pMNGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pM MSP(termed “MSP-600 pM).

FIG. 14-15 of WO2004/058184 are graphs showing that anti-NGF antagonistantibody E3 or Fab E3 did not inhibit survival of E17 nodose neuronspromoted by BDNF, NT4/5 or LIF. Mouse anti-NGF antagonist antibody 911was also tested, and similar results were observed. Specifically, FIG.14 of WO2004/058184 is a graph showing comparison of the effect ofvarious concentrations (200 nM or 80 nM) of full antibody E3 (termed “3Ein the figure”), Fab E3, full antibody 911, or Fab 911 on the survivalof E17 nodose neurons in the presence of no added neurotrophins (termed“no factors”), 400 pM BDNF (termed “BDNF-400 pM), 400 pM NT4/5 (termed“NT4/5-400 pM), or 2.5 nM LIF (termed “LIP-2.5 nM). FIG. 15 ofWO2004/058184 is a graph showing comparison of the effect of variousconcentrations (200 nM, 20 nM, 2 nM) of Fab E3 (termed “3E in thefigure”), or Fab 911 on the survival of E17 nodose neurons in thepresence of no added neurotrophins (termed “control”), 400 pM BDNF(termed “BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nMLIF (termed “LIP-2.5 nM).

Example 4 Preparation of Mammalian Expression Vectors and Expression ofAntibody E3 in Mammalian Cells

Three mammalian expression vectors were designed and constructed for usein the expression of antibody E3 in mammalian cells.

Vector Db.911.3E is an expression vector comprising the heavy chainvariable region of the E3 antibody and the human IgG2a constant region,and is suitable for transient or stable expression of the heavy chain.Db.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); a synthetic intron (nucleotides 619-1507); the DHFRcoding region (nucleotides 707-1267); human growth hormone signalpeptide (nucleotides 1525-1602); antibody 3E heavy chain variable region(nucleotides 1603-1965); human heavy chain IgG2a constant regioncontaining the following mutations: A330P331 to S330S331 (amino acidnumbering with reference to the wildtype IgG2a sequence; see Eur. J.Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal(nucleotides 2974-3217); SV40 enhancer region (nucleotides 3218-3463);phage f1 region (nucleotides 3551-4006) and beta lactamase (AmpR) codingregion (nucleotides 4443-5300). Db.911.3E was deposited at the ATCC onJan. 8, 2003, and was assigned ATCC Accession No. PTA-4895.

Vector Eb.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa chain constantregion, and is suitable for transient expression of the light chain.Eb.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); humangrowth hormone signal peptide (nucleotides 1173-1150); antibody E3 lightchain variable region (nucleotides 1251-1571); human kappa chainconstant region (nucleotides 1572-1892); SV40 late polyadenylationsignal (nucleotides 1910-2153); SV40 enhancer region (nucleotides2154-2399); phage f1 region (nucleotides 2487-2942) and beta lactamase(AmpR) coding region (nucleotides 3379-4236). Eb.911.3E was deposited atthe ATCC on Jan. 8, 2003, and was assigned ATCC Accession No. PTA-4893.

Vector Eb.pur.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa constant region,and is suitable for stable expression of the light chain. Eb.pur.911.3Econsists of nucleotide sequences corresponding to the following regions:the murine cytomegalovirus promoter region (nucleotides 1-612); humanEF-1 intron (nucleotides 619-1758); pac gene (puromycinR) coding region(nucleotides 739-1235); human hsp70 5′UTR region (nucleotides1771-1973); human growth hormone signal peptide (nucleotides 1985-2062);antibody E3 light chain variable region (nucleotides 2063-2383); humankappa chain constant region (nucleotides 2384-2704); SV40 latepolyadenylation signal (nucleotides 2722-2965); SV40 enhancer region(nucleotides 2966-3211); phage f1 region (nucleotides 3299-3654) andbeta lactamase (AmpR) coding region (nucleotides 4191-5048).Eb.pur.911.E3 was deposited at the ATCC on Jan. 8, 2003, and wasassigned ATCC Accession No. PTA-4894.

Transient cell expression was performed as follows: CHO and HEK293Tcells in 150 mm dishes were transiently co-transfected with 25 ug ofeach plasmid (i.e., one plasmid containing the heavy chain and oneplasmid containing the light chain). DNA was mixed with 100 ullipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. The DNA-lipid complexes were allowed to contact the cellsin DMEM/F12 medium without serum or antibiotics for 5 hours. Followingthis incubation, the media was changed for expression to Opti-MEM(Invitrogen) without any additives for two days. Cell supernatantscontaining antibody were harvested sequentially up to four times withsubsequent media replacement. Supernatants were purified by affinitychromatography using MapSelect Protein A resin (Amersham biosciences17-5199-02). Antibody was bound to the protein A resin in 0.3M glycine,0.6M NaCl buffer at pH 8, then eluted with 0.1 M citrate buffer at pH 3.Fractions containing antibody were immediately neutralized with 1M Trisbuffer at pH 8.0, Antibody fractions were then dialyzed and concentratedin PBS.

Example 5 Clinical Study

Patients with clinical diagnosis of interstitial cystitis are treatedwith anti-NGF antagonist antibody E3 to confirm the safety and efficacyof anti-NGF antibody E3 in treating one or more pain and lower urinarytract symptoms associated with interstitial cystitis and/or painfulbladder and/or bladder pain syndrome. The patients to be treated willhave moderate to severe interstitial cystitis as defined by a PelvicPain and Urgency/Frequency (PUF—Parsons et al. Urology 2002;60:573-578)) score of at least 16 and an O'Leary-Sant InterstitialCystitis Symptom Index (ICSI—O'Leary et al. Urology 1997; 49(Suppl5A):58-63) score of at least 8 at pre-treatment screening. Otherinclusion criteria are to be met. Patients who do not meet all of theinclusion criteria or meet one or more exclusion criteria will beconsidered a screen failure and will not be randomized into the study.

The efficacy of a single dose of the antibody via infusion compared to aplacebo in the treatment in pain and lower urinary tract symptomsassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome is evaluated. The E3 antibody isadministered intravenously to the patients at a dosage of 200 μg/kg. Inaddition to efficacy, the magnitude of effect of treatment compared withplacebo; the response of biomarkers to interstitial cystitis totreatment with the antibody; and the treatment response rate isevaluated, amongst other measurements. The biomarkers include NGF,glycoprotein-51 (GP-51), antiproliferative factor (APF) and HB-Epidermalgrowth factor (HB-EGF), amongst others.

The primary endpoint is analysed 6 weeks after intravenous infusion ofthe single dose of E3 antibody. Assessments at 2, 10 and 16 weekspost-dose are included as secondary endponts. The primary endpoint willbe a change in an 11-point pain numerical rating scale (NCS).O'Leary-Sant Interstitial Cystitis Symptom Index (ICSI) score is used asa secondary endpoint. Other secondary endpoints include O'Leary-SantInterstitial Cystitis Problem Index (ICPI—O'Leary et al. Urology 1997;49(Suppl 5A):58-63), ICSI plus ICPI, Pelvic Pain and Urgency/Frequency(PUF), average daily pain, micturition diary variables recorded by thepatient during the study period including including micturitionfrequency, nocturnal frequency, incontinence episode frequency, meanvolume voided per micturition, mean interstitial cystitis pain severity,urinary urgency episodes, average sleep disturbance score, average painassociated with sexual activity pain score, global response assessment,patent reporting treatment impact assessment, treatment failure,biomarkers, safety endpoints and/or pharmacokinetic measures. Biomarkersmay include NGF, glycoprotein-51 (GP-51), antiproliferative factor (APF)and HB-Epidermal growth factor (HB-EGF), amongst others.

The following data are derived from an interim analysis of an ongoingclinical trial utilising the above protocol to evaluate the safety andefficacy of E3 antibody in patients with interstitial cystitis/painfulbladder syndrome/bladder pain syndrome (IC/PBS/BPS). Data from 24patients was used in the interim analysis. A clear improvement(reduction) is seen in both the pain NRS and ICSI scores. The observedimprovement in patients receiving E3 antibody is greater than that seenin patients receiving placebo. These data provide support for the factthat E3 antibody may be effective in the treatment of the pain and otherlower urinary tract symptoms (LUTS) associated with IC/PBS/BPS. However,the final study result will need to be evaluated.

E3 antibody 200 mcg/kg Placebo Change from baseline to Week 6 in averagepain intensity N 13 11 LS Mean* (90% CI) −1.6 (−5.1, 1.8) −0.9 (−4.1,2.2) Difference vs Placebo* −0.7 (−2.3, 0.9) Change from baseline toWeek 6 in ICSI total score N 11  9 LS Mean*(90% CI)  −3.9 (−11.5, 3.6)−2.0 (−8.9, 4.9) Difference vs Placebo* −1.9 (−5.6, 1.7)

Pain was assessed using an 11 point (0-10) numerical rating scale (NRS).This scale is validated and widely used in the assessment of treatmentsdirected towards the relief of pain.

The Interstitial Cystitis Symptom Index (ICSI) assesses global ICsymptom severity, with scores ranging from 0 to 20. This is based on 4questions relating to pain and LUTS associated with IC/PBS/BPS. Theseinclude measurement of the severity of day and night-time urinaryfrequency, urinary urgency, and bladder pain over the preceding 4 weeks.

Deposit of Biological Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC):

ATCC Date of Material Accession No. Deposit Eb.911.3E E3 light chainPTA-4893 Jan. 8, 2003 Eb.pur.911.3E E3 light chain PTA-4894 Jan. 8, 2003Db.911.3E E3 heavy PTA-4895 Jan. 8, chain 2003

Details concerning these deposits can be found in WO2004058184, thecontent of which is herein incorporated by reference in its entirety.

Antibody sequences Heavy chain variable region (Kabat CDRs are under-lined; Chothia CDRs are BOLD AND ITALICIZED) (SEQ ID NO: 1)QVQLQESGPGLVKPSETLSLTCTVSGFSLI

WIRQPPGKGLEWIG

RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTLV TVS Light chain variable region (Kabat CDRs are under-lined; Chothia CDRs are BOLD AND ITALICIZED) (SEQ ID NO: 2)DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC

FGQGTKLEIKRT E3 heavy chain extended CDRs (SEQ ID NO: 3)CDRH1: GFSLIGYDLN (SEQ ID NO: 4) CDRH2: IIWGDGTTDYNSAVKS (SEQ ID NO: 5)CDRH3: GGYWYATSYYFDY E3 light chain extended CDRs (SEQ ID NO: 6)CDRL1: RASQSISNNLN (SEQ ID NO: 7) CDRL2: YTSRFHS (SEQ ID NO: 8)CDRL3: QQEHTLPYT Mouse monoclonal antibody 911 extended CDRs911 heavy chain extended CDRs (SEQ ID NO: 9) CDRH1: GFSLIGYDIN(SEQ ID NO: 10) CDRH2: MIWGDGTTDYNSALKS (SEQ ID NO: 11)CDRH3: GGYYYGTSYYFDY 911 light chain extended CDRs (SEQ ID NO: 12)CDRL1: RASQDISNHLN (SEQ ID NO: 13) CDRL2: YISRFHS (SEQ ID NO: 14)CDRL3: QQSKTLPYT E3 heavy chain amino acid sequence (full)(SEQ ID NO: 16) QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3E light chain amino acid sequence (full antibody) (SEQ ID NO: 17)DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC3E heavy chain nucleotide sequence (full antibody) (SEQ ID NO: 65)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCACCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAA3E heavy chain variable domain nucleotide sequence (SEQ ID NO: 66)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGT CACCGTCTCCTCA3E light chain nucleotide sequence (full antibody) (SEQ ID NO: 67)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTTCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACMAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA3E light chain variable domain nucleotide sequence (SEQ ID NO: 68)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGC

The above sequences and other sequences described herein are provided inthe attached sequence listing.

Sequence Listing Free Text

Sequences identified by SEQ ID Nos. 1-8, 15-68, 70-72, 74-77 in theattached sequence listing have the free text “synthetic construct”.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

The invention claimed is:
 1. A method for treating urinary urgencyassociated with interstitial cystitis and/or painful bladder syndromeand/or bladder pain syndrome in a subject, comprising administering aneffective amount of an anti-NGF antagonist antibody, thereby reducingtotal O'Leary-Sant Interstitial Cystitis Symptom Index (ICSI) score. 2.A method for treating urinary urgency associated with interstitialcystitis and/or painful bladder syndrome and/or bladder pain syndrome ina subject, comprising administering an effective amount of an anti-NGFantibody to the subject which: (a) binds NGF with a K_(D) of less thanabout 2 nM; (b) inhibits human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 of about 100 pM or less, wherein theIC50 is measured in the presence of about 15 pM human NGF; and (c)inhibits human NGF-dependent survival of mouse E13.5 trigeminal neuronswith an IC50 of about 10 pM or less, wherein the IC50 is measured in thepresence of about 1.5 pM of NGF, thereby reducing total O'Leary-SantInterstitial Cystitis Symptom Index (ICSI) score.
 3. A method fortreating urgency associated with interstitial cystitis and/or painfulbladder syndrome and/or bladder pain syndrome in a subject, comprisingadministering an effective amount of an anti-NGF antagonist antibodycomprising a heavy chain variable region comprising: (a) a CDR1 regionshown in SEQ ID NO: 3; (b) a CDR2 region shown in SEQ ID NO:4; and (c) aCDR3 region shown in SEQ ID NO:5; and a light chain variable regioncomprising: (a) a CDR1 region shown in SEQ ID NO: 6; (b) a CDR2 regionshown in SEQ ID NO:7; and (c) a CDR3 region shown in SEQ ID NO:8,wherein the antibody binds specifically to NGF, thereby reducing totalO'Leary-Sant Interstitial Cystitis Symptom Index (ICSI) score.
 4. Themethod of any one of claims 1-3, wherein the subject is a human.
 5. Themethod of claim 1 or 2, wherein the antibody is a monoclonal antibody.6. The method of claim 1 or 2, wherein the antibody is a humanizedantibody.
 7. The method of claim 1 or 2, wherein the antibody bindshuman NGF.